DC: Targeting Toxic Extrasynaptic Signaling in HD and AD

In neuronal transmission, as in real estate, location is everything.
When N-methyl D-aspartate (NMDA) glutamate receptors sit in the synapse,
they behave like good citizens, activating signaling pathways that
promote memory formation and neuron survival. However, when the
receptors loiter in the boondocks, they set off toxic signals that
damage synapses and kill neurons. Extrasynaptic receptors fire
particularly strongly in conditions like ischemia, Huntington’s disease
(HD), and Alzheimer’s disease (AD), making them a promising therapeutic
target (see Hardingham and Bading, 2010).
In a symposium at the Society for Neuroscience annual meeting, held
12-16 November 2011 in Washington, DC, scientists discussed some of the
latest research in this field. Session chair Stuart Lipton, from
the Sanford-Burnham Medical Research Institute, La Jolla, California,
pointed out that synaptic and extrasynaptic NMDA receptors vary in
several ways, including their downstream targets, their subunit
composition, and their firing patterns. Synaptic receptors fire in
bursts, while extrasynaptic ones are continuously active. Speakers
discussed these features in detail, as well as how extrasynaptic
signaling might be targeted in HD and AD.

Synaptic and extrasynaptic NMDA receptors trigger antagonistic signaling pathways, emphasized Hilmar Bading
at the University of Heidelberg, Germany. Synaptic signaling increases
calcium levels in the nucleus, activates the transcription factor CREB,
and leads to long-term learning and neuroprotection. By contrast,
extrasynaptic signals shut off CREB, initiate apoptotic pathways, and
cause breakdown of mitochondrial membranes. Bading’s group previously
showed that the two types of NMDA signaling turn on distinct programs of
gene expression (see ARF related news story on Zhang et al., 2007).
Nuclear calcium may act as the master switch that controls adaptive
responses such as neuronal plasticity and survival, Bading suggested. Of
the nearly 200 genes regulated by nuclear calcium, about 10 of them
make up the core neuroprotection program, Bading reported, and might be
useful therapeutically. One of these, the transcription factor Atf3,
reduces ischemic cell death by half in mice, Bading said.

Extrasynaptic NMDA receptors also vary from synaptic ones in
their composition. All NMDA receptors consist of two GluN1 and two GluN2
subunits, but GluN2 subunits come in different forms. In the mature
brain, GluN2A subunits predominate in synapses, while GluN2B
preferentially populates extrasynaptic sites. This partitioning suggests
that GluN2B plays the major role in excitotoxicity. The subunits also
possess distinct C-terminal, cytoplasmic tails, allowing them to
interact with different intracellular proteins. Interestingly, in early
development, when GluN2B dominates at all NMDA receptor sites, synaptic
signaling protects neurons from cell death, indicating that the GluN2B
subunit does not necessarily activate cell death pathways (see Martel et al., 2009). Nonetheless, Giles Hardingham
at the University of Edinburgh, U.K., wondered if subunit composition
could help explain the differing toxicities of synaptic and
extrasynaptic receptors.

To look more closely at the issue, Hardingham and colleagues made
chimeric constructs, replacing the tail of GluN2B with that of 2A.
Cultured neurons containing the chimeric receptor resisted
excitotoxicity better than wild-type cells, Hardingham reported.
Signaling by the GluN2B tail does play a role in harming cells,
Hardingham concluded, and this difference is most noticeable in the
context of a mild excitotoxic insult. Why is synaptic GluN2B less toxic
than extrasynaptic? Hardingham noted that GluN2B toxicity only becomes
apparent during chronic activation, such as that found in extrasynaptic
sites. Looking for the downstream mechanism behind GluN2B
excitotoxicity, Hardingham found that GluN2B signaling represses CREB,
an important neuroprotective factor.

The difference between GluN2A and GluN2B excitotoxicity might have consequences for disease. Lynn Raymond
at the University of British Columbia, Vancouver, pointed out that
GluN2B is enriched in GABAergic medium spiny neurons of the striatum,
which selectively degenerate in HD (see Raymond et al., 2011).
Degeneration probably occurs through an excitotoxic mechanism, Raymond
noted, as injecting glutamate and NMDA into the striatum can reproduce
the symptoms of HD in mice. Raymond’s group found that HD mice have more
extrasynaptic receptors than do wild-type mice (see ARF related news story on Milnerwood et al., 2010). In addition, overexpressing GluN2B in an HD mouse causes more atrophy, Raymond said.

Raymond compared HD mice (YAC128), which express mutant
huntingtin protein containing 128 CAG repeats, with control mice that
have normal huntingtin with 18 repeats. She reported that the YAC128
mice have more GluN2B at the cell surface than do controls, and that
most of it is extrasynaptic. Looking for the mechanism, she found that
the scaffold protein PSD-95, which stabilizes and maintains GluN2B in
synaptic sites, shifts to extrasynaptic locales in HD mice. PSD-95 binds
directly to huntingtin protein, and binds more tightly to the mutant
form, Raymond noted, which may be linked to the relocation. In addition,
striatal-enriched tyrosine phosphatase (STEP) is more active in HD mice
than in wild-type. STEP dephosphorylates GluN2B, causing it to abandon
the synapse and potentially freeing it up for incorporation into
extrasynaptic receptors. Inhibiting STEP increases synaptic GluN2B,
Raymond said. Downstream of GluN2B, Raymond found that the
mitogen-activated protein kinase p38 was more active in the HD mice, and
inhibiting it protected cultured HD neurons from death, suggesting that
this protein could be a therapeutic target.

One sign of the importance of extrasynaptic signaling in HD is
that memantine, an approved AD drug that selectively silences
extrasynaptic signaling (see ARF related news story on Xia et al., 2010), reverses motor learning deficits in HD mice (see Okamoto et al., 2009). A UBC group led by Blair Leavitt is currently testing memantine in HD patients in a Phase 2 trial.

Memantine relieves symptoms in moderate AD, probably by dampening
extrasynaptic signaling. In his talk, Lipton turned to the role that
this type of signaling plays in AD. Recent work showed that Aβ can
inhibit synaptic glutamate reuptake, causing the neurotransmitter to
spill over and activate extrasynaptic sites (see ARF related news story on Li et al., 2011).
Lipton reported that oligomeric Aβ also induces cultured astrocytes to
spit out more glutamate. Lipton noted that this finding dovetails with
data presented by Annalisa Scimemi at the National Institute of
Neurological Disorders and Stroke, Bethesda, Maryland, in a separate SfN
session. The most abundant glutamate transporter in adult brain, GLT-1,
is found mostly in astrocytes, and is responsible for mopping up
extracellular glutamate. Scimemi reported that adding synthetic Aβ42
(oligomeric and monomeric) to hippocampal slices increased deposits of
insoluble GLT-1 and doubled the time course of glutamate clearance by
the transporter. This provides yet another mechanism behind elevated
glutamate in AD brains.

Lipton, an author on worldwide memantine patents, said that the
drug improves AD symptoms by acting as a low-affinity, transient NMDA
channel blocker. This helps “turn down the volume” on NMDA transmission
without silencing it, Lipton said. In addition, memantine blocks only
open channels, allowing the drug to selectively block extrasynaptic
receptors, which are chronically active, while largely sparing synaptic
sites. However, memantine is not effective enough, Lipton said. He is
developing a new version, nitro-memantine, that is even more selective
for extrasynaptic sites. In addition to having the same effect as
memantine in the NMDA receptor ion channel, nitro-memantine also
nitrosylates the NMDA receptor, i.e., it transfers a nitric oxide group
to a cysteine residue on the receptor, which desensitizes the channel.
Lipton tested the drug in hippocampal slices from transgenic APP mice (J20),
which show chronic extrasynaptic activity. In these mice, synaptic
spine density is half that in wild-type mice. While memantine protects
some spines, nitro-memantine restored spine density to virtually normal
levels. Moving to an in-vivo system, Lipton reported that when 3xTg mice
were treated with nitro-memantine for three months, levels of
synaptophysin, a marker of synapses, returned to normal, and the mice
improved in an object exploration test. In contrast, when treated with
memantine, the mice showed no behavioral improvement. Lipton hopes the
greater efficacy of nitro-memantine will eventually translate to people,
although clinical trials are not yet scheduled.

Synaptic NMDA signaling not only protects neurons from insults,
but also enables the encoding of long-term memories. In his talk, Bading
noted that nuclear calcium may play a crucial role in learning, perhaps
through the action of CREB, a key memory protein (see, e.g., ARF related news story).
Using transgenic flies that express a neuronal calcium sensor, Bading
found that calcium floods into the nucleus during learning. When he
activated CaMBP4, an inhibitor of nuclear calcium signaling, the flies’
ability to remember an association 24 hours after learning dropped
dramatically. Another gene stimulated by calcium signaling is vascular
endothelial growth factor D (VEGF-D). Bading reported that VEGF-D
maintains dendrite length and complexity, and is necessary for long-term
memory in mice (see ARF related news story on Mauceri et al., 2011).
Nuclear calcium signaling is not beneficial in all contexts, however.
Blocking nuclear calcium signaling in the spinal cord dampens chronic
inflammatory pain, Bading noted.


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