| Reference | 1. Front Pharmacol. 2016 Dec 12;7:486. doi: 10.3389/fphar.2016.00486. eCollection
2016.
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Pharmacological Characterization of the Native Store-Operated Calcium Channels of
Cortical Neurons from Embryonic Mouse Brain.
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In the murine brain, the first post-mitotic cortical neurons formed during
embryogenesis express store-operated channels (SOCs) sensitive to Pyr3, initially
proposed as a blocker of the transient receptor potential channel of C type 3
(TRPC3 channel). However, Pyr3 does not discriminate between Orai and TRPC3
channels, questioning the contribution of TRPC3 in SOCs. This study was
undertaken to clarify the molecular identity and the pharmacological profile of
native SOCs from E13 cortical neurons. The mRNA expression of STIM1-2 and Orai1-3
was assessed by quantitative reverse transcription polymerase chain reaction. E13
cortical neurons expressed STIM1-2 mRNAs, with STIM2 being the predominant
isoform. Only transcripts of Orai2 were found but no Orai1 and Orai3 mRNAs.
Blockers of Orai and TRPC channels (Pyr6, Pyr10, EVP4593, SAR7334, and GSK-7975A)
were used to further characterize the endogenous SOCs. Their activity was
recorded using the fluorescent Ca2+ probe Fluo-4. Cortical SOCs were sensitive to
the Orai blockers Pyr6 and GSK-7975A, as well as to EVP4593, zinc, copper, and
gadolinium ions, the latter one being the most potent SOCs blocker tested (IC50
～10 nM). SOCs were insensitive to the TRPC channel blockers Pyr10 and SAR7334. In
addition, preventing mitochondrial Ca2+ uptake inhibited SOCs which were
unaffected by inhibitors of the Ca2+-independent phospholipase A2. Altogether,
Orai2 channels are present at the beginning of the embryonic murine
cortico-genesis and form the core component of native SOCs in the immature
cortex. This Ca2+ route is likely to play a role in the formation of the brain
cortex.
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2. Mol Neurodegener. 2016 Apr 14;11:27. doi: 10.1186/s13024-016-0092-5.
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Manifestation of Huntington/’s disease pathology in human induced pluripotent stem
cell-derived neurons.
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BACKGROUND: Huntington/’s disease (HD) is an incurable hereditary
neurodegenerative disorder, which manifests itself as a loss of GABAergic medium
spiny (GABA MS) neurons in the striatum and caused by an expansion of the CAG
repeat in exon 1 of the huntingtin gene. There is no cure for HD, existing
pharmaceutical can only relieve its symptoms.
RESULTS: Here, induced pluripotent stem cells were established from patients with
low CAG repeat expansion in the huntingtin gene, and were then efficiently
differentiated into GABA MS-like neurons (GMSLNs) under defined culture
conditions. The generated HD GMSLNs recapitulated disease pathology in vitro, as
evidenced by mutant huntingtin protein aggregation, increased number of
lysosomes/autophagosomes, nuclear indentations, and enhanced neuronal death
during cell aging. Moreover, store-operated channel (SOC) currents were detected
in the differentiated neurons, and enhanced calcium entry was reproducibly
demonstrated in all HD GMSLNs genotypes. Additionally, the quinazoline
derivative, EVP4593, reduced the number of lysosomes/autophagosomes and SOC
currents in HD GMSLNs and exerted neuroprotective effects during cell aging.
CONCLUSIONS: Our data is the first to demonstrate the direct link of nuclear
morphology and SOC calcium deregulation to mutant huntingtin protein expression
in iPSCs-derived neurons with disease-mimetic hallmarks, providing a valuable
tool for identification of candidate anti-HD drugs. Our experiments demonstrated
that EVP4593 may be a promising anti-HD drug.
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3. J Neurosci. 2016 Jan 6;36(1):125-41. doi: 10.1523/JNEUROSCI.1038-15.2016.
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Enhanced Store-Operated Calcium Entry Leads to Striatal Synaptic Loss in a
Huntington/’s Disease Mouse Model.
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In Huntington/’s disease (HD), mutant Huntingtin (mHtt) protein causes striatal
neuron dysfunction, synaptic loss, and eventual neurodegeneration. To understand
the mechanisms responsible for synaptic loss in HD, we developed a
corticostriatal coculture model that features age-dependent dendritic spine loss
in striatal medium spiny neurons (MSNs) from YAC128 transgenic HD mice.
Age-dependent spine loss was also observed in vivo in YAC128 MSNs. To understand
the causes of spine loss in YAC128 MSNs, we performed a series of mechanistic
studies. We previously discovered that mHtt protein binds to type 1 inositol
(1,4,5)-trisphosphate receptor (InsP3R1) and increases its sensitivity to
activation by InsP3. We now report that the resulting increase in steady-state
InsP3R1 activity reduces endoplasmic reticulum (ER) Ca(2+) levels. Depletion of
ER Ca(2+) leads to overactivation of the neuronal store-operated Ca(2+) entry
(nSOC) pathway in YAC128 MSN spines. The synaptic nSOC pathway is controlled by
the ER resident protein STIM2. We discovered that STIM2 expression is elevated in
aged YAC128 striatal cultures and in YAC128 mouse striatum. Knock-down of InsP3R1
expression by antisense oligonucleotides or knock-down or knock-out of STIM2
resulted in normalization of nSOC and rescue of spine loss in YAC128 MSNs. The
selective nSOC inhibitor EVP4593 was identified in our previous studies. We now
demonstrate that EVP4593 reduces synaptic nSOC and rescues spine loss in YAC128
MSNs. Intraventricular delivery of EVP4593 in YAC128 mice rescued age-dependent
striatal spine loss in vivo. Our results suggest EVP4593 and other inhibitors of
the STIM2-dependent nSOC pathway as promising leads for HD therapeutic
development.SIGNIFICANCE STATEMENT: In Huntington/’s disease (HD) mutant
Huntingtin (mHtt) causes early corticostriatal synaptic dysfunction and eventual
neurodegeneration of medium spine neurons (MSNs) through poorly understood
mechanisms. We report here that corticostriatal cocultures prepared from YAC128
HD mice feature age-dependent MSN spine loss, mirroring YAC128 MSN spine loss in
vivo. This finding establishes a system for mechanistic studies of synaptic
instability in HD. We use it to demonstrate that sensitization of type 1 inositol
(1,4,5)-trisphosphate receptors by mHtt, which depletes endoplasmic reticulum
calcium, contributes to synaptotoxic enhancement of STIM2-dependent
store-operated calcium (SOC) entry. Treatment with EVP4593, a neuroprotective
inhibitor of neuronal SOC channels, rescues YAC128 MSN spine loss both in vitro
and in vivo. These results suggest that enhanced neuronal SOC causes synaptic
loss in HD-afflicted MSNs.
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4. Front Physiol. 2015 Nov 24;6:337. doi: 10.3389/fphys.2015.00337. eCollection
2015.
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Both Orai1 and TRPC1 are Involved in Excessive Store-Operated Calcium Entry in
Striatal Neurons Expressing Mutant Huntingtin Exon 1.
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It has been previously reported that N-terminus of mutant huntingtin (product of
the 1st exon) is sufficient to cause a Huntington/’s disease (HD) pathological
phenotype. In view of recent data suggesting that improper regulation of
store-operated calcium (SOC) channels is involved in neurodegenerative processes,
we investigated influence of expression of the mutant huntingtin N-terminal
fragment (Htt138Q-1exon) on SOC entry (SOCE) in mouse neuroblastoma cells
(Neuro-2a) and in primary culture of medium spiny neurons (MSNs) isolated from
mice. The results show that SOCE in these cells is enhanced upon lentiviral
expression of the Htt138Q-1exon. Moreover, we demonstrated that RNAi-mediated
knockdown of TRPC1, Orai1, or STIM1 proteins leads to dramatic reduction of
abnormal SOCE in both Neuro-2a and MSNs, expressing Htt138Q-1exon. Thus, we
concluded that abnormal SOCE in these cells is maintained by both TRPC1- and
Orai1-containing channels and required STIM1 for its activation. Furthermore,
EVP4593 compound previously tested as a potential anti-HD drug in a Drosophila
screening system has proved to be capable of reducing SOCE to the normal level in
MSNs expressing the Htt138Q-1exon.
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