Download embj201488977-sup-0010-Suppl

Romanov, Alpar et al.
Date of submission: 06/05/2017
EMBOJ-2014-88977; revised submission
Supplementary Information to the manuscript:
A secretagogin locus of the mammalian hypothalamus controls
stress hormone release
Roman Romanov*, Alan Alpar*,#, Ming-Dong Zhang, Amit Zeisel, André
Calas, Marc Landry, Matthew Fuszard, Sally L. Shirran, Robert Schnell,
Arpad Dobolyi, Mark Olah, Lauren Spence, Henrik Marten, Miklos
Palkovits, Matthias Uhlen, Harald H. Sitte, Catherine M. Botting, Ludwig
Wagner, Sten Linnarsson, Tomas Hökfelt++ & Tibor Harkany++,#
*R.R. & A.A. contributed equally to this study.
++T.Hö.
& T.Ha. share senior authorship.
#Address
correspondence to:
Tibor Harkany (at the Karolinska
Institutet); Telephone: +46 8 524 87656; Fax: +46 8
341 960; e-mail: [email protected] or Alan Alpar (at
the
Semmelweis
University);
e-mail:
[email protected]
This file contains:
Supplementary Figures 1 - 6
Supplementary Tables 1 - 3
Legends to Supplementary Figures & Tables
Supplementary References
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Romanov, Alpar et al.
Date of submission: 06/05/2017
EMBOJ-2014-88977; revised submission
Legends to Supplementary Tables & Figures
Suppl. Table 1
Electrophysiological parameters used to sub-classify PVN neurons in
ex vivo brain slice preparations.
Data are from: type Ia: n = 17; type Ib: n = 5; type IIa: n = 10; type IIb:
n = 7; type IIIa: n = 18; type IIIb: n = 16 neurons, and were expressed
as means ± s.e.m. Parameters were chosen according to published
protocols(Miyoshi et al, 2010). Sub-clustering was based on the posthoc identification of biocytin-filled neurons using AVP/oxytocin
(combined in triple-labeling experiments and used as “magnocellular
markers”) and secretagogin (scgn) as histochemical markers (see
also Fig. 3A,B).
Suppl. Table 2
List of primary antibodies used for multiple immunofluorescence
labeling.
All antibodies were affinity purified, and extensively tested in vitro
and in vivo, including relevant genetic controls (Benoit et al, 1980;
Mulder et al, 2009; Hartig et al, 2009; Westberg et al, 2009;
Dabrowska et al, 2011; Stanic et al, 2011; Attems et al, 2012; Shi et
al, 2012; Renner et al, 2012; Keimpema et al, 2013).
Suppl. Table 3
Proteins identified by differential interactome analysis of Ca 2+-bound
secretagogin.
A list of 97 proteins that were found when immunoprecipitation (IP) of
secretagogin was performed in the presence of 10 µM Ca2+. Criteria
for positive protein identification are shown at the top. Proteins were
classified by color coding to correspond to the cumulative data
shown in Fig. 5C1. Non-targeted IgG in the presence of 10 µM Ca2+
was used as negative control. Proteins (n = 15 in total) marked under
“anti-secretagogin IP, Ca2+-free” might have some ability to
constitutively interact with secretagogin. Note that the number of
peptides fragments is low, likely reflecting the sparsity of proteinprotein interactions under Ca2+-free conditions.
Suppl. Figure 1
Electrophysiological classification of PVN neurons.
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Romanov, Alpar et al.
Date of submission: 06/05/2017
EMBOJ-2014-88977; revised submission
Criteria were chosen as per (Luther et al, 2000; Stern, 2001; Luther
et al, 2002; Wamsteeker Cusulin et al, 2013). Example traces are
known for each neuronal subgroup. Statistically-analyzed data are
presented in Supplementary Table 1.
Suppl. Figure 2
Secretagogin-expressing
PVN
neurons
exhibit
parvocellular
identities, including dendrite morphology.
(A) Variations of mRNA transcript levels in cells dissociated from the
mouse PVN. The x-axis is scaled to show only a fraction of nonexpressing neurons (at 0 levels) for visual clarity. Red circles
correspond to secretagogin+ neurons. Horizontal dashed lines show
the cut-off value used to scale positive neurons for each mRNA
transcript.
(B-B2)
Morphometric
secretagogin+
reconstruction
(B1) and
of
vasopressin+
vasopressin+/secretagogin+
(B),
(B2) neurons
isolated from mouse hypothalamus at postnatal days (P)1-2. Scale
bar = 30 µm.
(C) Morphological parameters of cultured hypothalamic neurons
identified post-hoc by multiple immunofluorescence cytochemistry.
Secretagogin+ neurons had smaller somata, with a higher number of
processes that were longer and arborized more extensively than
those of vasopressin+ neurons (11.61 ± 0.72 vs. 9.04 ± 0,21, p <
0.05). Vasopressin+/secretagogin+ neurons were morphologically
similar to vasopressin+/secretagogin- neurons (12.31 ± 0.61 vs. 11.61
± 0.72).
(D)
Representative
intracellular
Ca2+
responses
(ratiometric
measurements by using Fura-2AM) from n ≥ 3 neurons/group
exposed to depolarizing conditions (55 mM KCl).
Suppl. Figure 3
The effect of secretagogin on pharmacologically-induced Ca2+
responses in PVN neurons.
(A) Representative image of cultured hypothalamic neurons loaded
with Fura-2AM (left panel) and post-hoc histochemistry for AVP and
secretagogin (scgn). Scale bar = 60 µm.
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Romanov, Alpar et al.
Date of submission: 06/05/2017
EMBOJ-2014-88977; revised submission
(B) Representative Ca2+ traces from cytochemically-identified PVN
neurons after ratiometric Fura-2AM imaging demonstrate the
existence of different response patterns to pharmacological stimuli.
Drug concentrations were as follows: 55 mM KCl, 50 µM glutamate,
50 µM NMDA + 100 µM glycine, 50 µM kainate, 50 µM carbachol, 10
µM ATP, 100 µM GABA and 50 µM bicuculline (Yamashita et al,
1987; Collden et al, 2010; Haam et al, 2012). Color-coded traces
correspond to responses from single cells.
(C) Cumulative (mean) responses per group upon depolarization by
55 mM KCl.
(D) Statistical analysis of Ca2+ responses, expressed as % change.
AVP+ (in blue; total n = 24), secretagogin+ (in red; total n = 69) and
other (unspecified, n = 14) neurons were analyzed; n = 14 - 69
neurons/group. ***p < 0.001; **p < 0.01; *p < 0.05; Student’s t-test.
Suppl. Figure 4
Effect of secretagogin overexpression on Ca2+ signaling in SH-SY5Y
human neuroblastoma cells.
(A) Overexpression of secretagogin in SH-SY5Y cells was used to
estimate the Ca2+-binding protein’s buffer capacity (B) in response to
excitatory stimuli as indicated.
(C) Immunofluorescence signal intensity (in green; A) was scaled
and correlated with Ca2+ responsiveness upon secretagogin
overexpression. Individual data points were presented. Dashed lines
were color-coded to identify specific stimuli and denote the lack of
correlation between Ca2+ peaks vs. secretagogin levels. Scale bar =
40 µm.
Suppl. Figure 5
In vivo siRNA-mediated secretagogin silencing does not affect
prominent hormone and neuropeptide systems in the mouse
paraventricular nucleus.
(A)
Arginine-vasopressin
(AVP)-immunoreactive
PVN
neurons
showed unchanged distribution in adult mice that had been injected
with secretagogin-specific siRNA in the paraventricular nucleus
(“scgn siRNA”) relative to control animals (“non-target siRNA”).
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Romanov, Alpar et al.
Date of submission: 06/05/2017
EMBOJ-2014-88977; revised submission
(B,C) Likewise, the distribution of both neuropeptide Y-containing
processes (B) and oxytocin-containing neurons (C) in the PVN was
found unperturbed in animals exposed to secretagogin-specific
siRNA.
(D) In the arcuate nucleus, which borders the ventral-most wall of the
3rd ventricle and therefore can be accessible to siRNA molecules,
somatostatin immunoreactivity was unaffected.
Abbreviations: 3V, 3rd ventricle; PVN, paraventricular nucleus of the
hypothalamus. Scale bars = 100 µm (A-D).
Suppl. Figure 6
Summary
cartoon
for
secretagogin
action
and
interactome
engagement in CRH neurons.
(A) Secretagogin can affect CRH release either indirectly, by
affecting the function of key proteins involved in the vesicle formation
and cargo along the axons to the median eminence (“vesicle
logistics”), or more directly, by Ca2+-dependent modulation of the
exocytosis machinery in situ in nerve terminals (“release site”). This
duality of action suggests a significant role for secretagogin in
defining the dynamics of Ca2+-dependent neuropeptide release.
(B) Proteome profiling-based secretagogin interactions likely to
control neuropeptide release. Select protein-protein interactions are
indicated.
Note
that
secretagogin’s
localization
along
the
plasmalemma suggests its ability to also modulate intra-membrane
adaptor proteins. So far, our analysis leaves ambiguity as to the
molecular identity of the exact identity of the Rab3 family member
modulated by secretagogin, henceforth “Rab3” labeling is used.
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Romanov, Alpar et al.
Date of submission: 06/05/2017
EMBOJ-2014-88977; revised submission
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EMBOJ-2014-88977; revised submission
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