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Interpericyte tunnelling nanotubes regulate neurovascular coupling

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Interpericyte tunnelling nanotubes regulate neurovascular coupling

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Signalling between cells of the neurovascular unit, or neurovascular coupling, is essential to match local blood flow with neuronal activity. Pericytes interact with endothelial cells and extend processes that wrap capillaries, covering up to 90% of their surface area1,2. Pericytes are candidates to regulate microcirculatory blood flow because they are strategically positioned along capillaries, contain contractile proteins and respond rapidly to neuronal stimulation3,4, but whether they synchronize microvascular dynamics and neurovascular coupling within a capillary network was unknown. Here we identify nanotube-like processes that connect two bona fide pericytes on separate capillary systems, forming a functional network in the mouse retina, which we named interpericyte tunnelling nanotubes (IP-TNTs). We provide evidence that these (i) have an open-ended proximal side and a closed-ended terminal (end-foot) that connects with distal pericyte processes via gap junctions, (ii) carry organelles including mitochondria, which can travel along these processes, and (iii) serve as a conduit for intercellular Ca²⁺ waves, thus mediating communication between pericytes. Using two-photon microscope live imaging, we demonstrate that retinal pericytes rely on IP-TNTs to control local neurovascular coupling and coordinate light-evoked responses between adjacent capillaries. IP-TNT damage following ablation or ischaemia disrupts intercellular Ca²⁺ waves, impairing blood flow regulation and neurovascular coupling. Notably, pharmacological blockade of Ca²⁺ influx preserves IP-TNTs, rescues light-evoked capillary responses and restores blood flow after reperfusion. Our study thus defines IP-TNTs and characterizes their critical role in regulating neurovascular coupling in the living retina under both physiological and pathological conditions.
IP-TNTs connect pericytes on distal capillaries in a soma-to-process configuration a–c, Pericyte extending a nanotube process, which we name an IP-TNT (dotted line), across an adjacent capillary. All retinas presented IP-TNTs. d, e, IP-TNT diameter (n = 86 IP-TNTs/pericytes, n = 3 mice; two-tailed Student’s t-test, NS, not significant) and length (n = 91 IP-TNTs/pericytes, n = 3 mice; two-tailed Mann–Whitney U test, ***P < 0.001); data are mean ± s.e.m. f, Incidence of short versus long IP-TNTs (n = 91 IP-TNTs/pericytes, n = 3 mice). g, h, Labelling of IP-TNTs with phalloidin (g) and lectin (h), showing F-actin cytoskeleton. i, Fluorescein electroporated into a pericyte with IP-TNT diffuses from soma and IP-TNT into distal pericyte (pipette in green; replicated five times). j, Pericyte with IP-TNT (inset), visualized by confocal imaging, is selected for FIB-SEM. k, Montage of FIB-SEM images of the pericyte in j (inset) highlighting its nucleus (purple), IP-TNT (yellow, dotted line), distal pericyte processes (dpp, red) and capillaries (green). l, Reconstruction of FIB-SEM images in k. m, Open-ended side of IP-TNT is continuous with the pericyte cytoplasm. n, Mitochondria within IP-TNT (left) and at its end-foot (ef; right). o–q, IP-TNTs contain vesicles (o) and endoplasmic reticulum (ER; p), and membrane-to-membrane contacts exist between the IP-TNT ef (yellow) and dpp (red) (q). Four hundred FIB-SEM sections of an IP-TNT were analysed. r, Top, CX43 immunostaining (green) in two IP-TNT-connected pericytes. Bottom, inset shows CX43 labelling (green) between the IP-TNT ef and the dpp. s–u, Fluorescein electroporated in the presence of carbenoxolone accumulates in proximal pericyte and IP-TNT (s, t, left), delineating contact between IP-TNT ef (green) and dpp (red) (u). Right, higher magnification of inset (boxed in s–u). v, Higher magnification of inset (boxed in u, right) shows localization of CX43-positive plaques (yellow) at interface between IP-TNT ef (green) and dpp (red) (replicated 12 times). The same IP-TNT-coupled pericytes in s–u were processed for CX43 labelling (replicated three times). Source data
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IP-TNTs are a conduit for pericyte-to-pericyte communication in living retinas a, Setup for two-photon laser scanning microscopy (TPLSM). b, In vivo imaging using intravenously applied FITC (to visualize capillaries) and intravitreally injected lectin (to label the basement membrane of pericytes) shows a network of IP-TNTs linking distal capillaries, observed in all retinas analysed. c, d, Short and long IP-TNTs exist in all vascular plexuses. e, Colabelling with phalloidin to detect F-actin (top) and lectin to visualize pericytes/IP-TNTs (bottom) shows that IP-TNTs contain an F-actin cytoskeleton. f, Time-lapse imaging shows mitochondria travelling within an IP-TNT (arrow; replicated 3 times). g, TPLSM recordings show intercellular Ca²⁺ wave (ICWs) propagating through an IP-TNT (dotted line; replicated in 57 IP-TNTs, n = 20 mice). h, ICWs are synchronous Ca²⁺ increases in IP-TNT-coupled pericyte pairs (grey). i, j, ICW frequency decreases with carbenoxolone (CBX) treatment (in j, vehicle: n = 18 capillaries, n = 4 mice; CBX: n = 16 capillaries, n = 4 mice; *P = 0.029). k, Longitudinal analysis of light-evoked changes in capillary diameter in vivo (blue, dilation, n = 39 capillaries, n = 2 mice; red, constriction, n = 30 capillaries, n = 2 mice; *P < 0.05, **P < 0.01). l, TPLSM quantification of red blood cells (RBC) per time unit after light stimulation (replicated in 224 capillaries, n = 28 mice). m, IP-TNT-linked capillaries undergo opposite blood flow responses (dilation: n = 27 capillaries, n = 2 mice; constriction: n = 10 capillaries, n = 2 mice; ***P < 0.001). n, ICWs in IP-TNT-linked capillaries before and after light stimulation (n = 13 capillaries per group, n = 5 mice per group; NS, not significant). o, p, A decrease in Ca²⁺ transient frequency correlates with dilation (pericyte 1), whereas increased frequency correlates with constriction in the coupled pericyte (pericyte 2). p shows Ca²⁺ transient frequency changes in IP-TNT-linked pericytes (n = 13 pericytes per group, n = 5 mice per group; **P < 0.01). q, r, Pericytes at constricting and dilating capillaries display increases and decreases in Ca²⁺ transient frequency, respectively (constriction: n = 10 pericytes, n = 5 mice; dilation: n = 16 pericytes, n = 5 mice, **P = 0.008). Data are mean ± s.e.m. j, m, n, two-tailed Mann–Whitney U test; k, two-tailed ANOVA Dunnett’s test; p–r, two-tailed Student’s t-test. Source data
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Ischaemia damages IP-TNTs and leads to microvascular dysfunction a, Retinal ischaemia induced by transient ligature of ophthalmic vessels. b, TPLSM imaging of pericytes and capillaries at 10 and 40 min after induction of ischaemia or sham surgery (replicated 3 times per group). c, TPLSM shows reduction in capillary diameter in ischaemia relative to sham controls (sham: n = 75 capillaries, n = 3 mice; ischaemia: n = 29 capillaries, n = 3 mice; **P < 0.01, ***P < 0.001). d, Time-lapse TPLSM imaging of IP-TNT (dotted line) disintegration during ischaemia (arrows; replicated 3 times). e, Ischaemia also ruptures IP-TNTs ex vivo. f, Increase in damaged IP-TNTs in ischaemia relative to controls (ischaemia: n = 6 mice; sham-operated: n = 4; *P = 0.038). g–i, IP-TNT damage induced by ischaemia–reperfusion causes loss of light-evoked capillary dilation (blue) and constriction (red) (pre-ischaemia: dilation, n = 21 capillaries, n = 3 mice, constriction, n = 21 capillaries, n = 3 mice; post-ischaemia: dilation, n = 18 capillaries, n = 4 mice, constriction, n = 21 capillaries, n = 5 mice). h, Capillary responses are impaired in ischaemia (dilation: sham, n = 46 capillaries, n = 3 mice, ischaemia–reperfusion (I–R), n = 20, n = 4 mice; constriction: sham, n = 60, n = 3 mice, reperfusion, n = 21, n = 5 mice; *P = 0.016, **P = 0.002). i, Blood flow is compromised in ischaemia–reperfusion (dilation: sham, n = 31 capillaries, n = 5 mice, reperfusion, n = 5 capillaries, n = 5 mice; constriction: sham, n = 28 capillaries, n = 5 mice, reperfusion, n = 10 capillaries, n = 5 mice; *P = 0.034, NS, not significant). j–m, Nifedipine restores light-evoked capillary responses (dilation: sham + nifedipine, n = 14 capillaries, n = 4, reperfusion + vehicle, n = 27 capillaries, n = 5, reperfusion + nifedipine, n = 17 capillaries, n = 4; constriction: sham + nifedipine, n = 20 capillaries, n = 4, reperfusion + vehicle, n = 21 capillaries, n = 5, reperfusion + nifedipine, n = 18 capillaries, n = 4; *P < 0.05, ***P < 0.001, NS, not significant). n, Blood flow is impaired after ischaemia–reperfusion, but restored by nifedipine (dilation: sham + nifedipine, n = 16 capillaries, n = 4, reperfusion + vehicle, n = 13 capillaries, n = 5, reperfusion + nifedipine, n = 12 capillaries, n = 4; constriction: sham + nifedipine, n = 10 capillaries, n = 4, reperfusion + vehicle, n = 11 capillaries, n = 5, reperfusion + nifedipine, n = 13 capillaries, n = 4; *P < 0.05, NS, not significant). Data are mean ± s.e.m. c, m, n, two-tailed ANOVA Tukey’s test; f, h, i, two-tailed Student’s t-test. Source data
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Nature | Vol 585 | 3 September 2020 | 91
Article
Interpericyte tunnelling nanotubes regulate
neurovascular coupling
Luis Alarcon-Martinez1,2,4 ✉, Deborah Villafranca-Baughman1,2,4, Heberto Quintero1,2,
J. Benjamin Kacerovsky3, Florence Dotigny1,2, Keith K. Murai3, Alexandre Prat1,2,
Pierre Drapeau1,2 & Adriana Di Polo1,2 ✉
Signalling between cells of the neurovascular unit, or neurovascular coupling, is
essential to match local blood ow with neuronal activity. Pericytes interact with
endothelial cells and extend processes that wrap capillaries, covering up to 90% of
their surface area1,2. Pericytes are candidates to regulate microcirculatory blood ow
because they are strategically positioned along capillaries, contain contractile
proteins and respond rapidly to neuronal stimulation3,4, but whether they
synchronize microvascular dynamics and neurovascular coupling within a capillary
network was unknown. Here we identify nanotube-like processes that connect two
bona de pericytes on separate capillary systems, forming a functional network in the
mouse retina, which we named interpericyte tunnelling nanotubes (IP-TNTs). We
provide evidence that these (i) have an open-ended proximal side and a closed-ended
terminal (end-foot) that connects with distal pericyte processes via gap junctions, (ii)
carry organelles including mitochondria, which can travel along these processes, and
(iii) serve as a conduit for intercellular Ca2+ waves, thus mediating communication
between pericytes. Using two-photon microscope live imaging, we demonstrate that
retinal pericytes rely on IP-TNTs to control local neurovascular coupling and
coordinate light-evoked responses between adjacent capillaries. IP-TNT damage
following ablation or ischaemia disrupts intercellular Ca2+waves, impairing blood
ow regulation and neurovascular coupling. Notably, pharmacological blockade of
Ca2+ inux preserves IP-TNTs, rescues light-evoked capillary responses and restores
blood ow after reperfusion. Our study thus denes IP-TNTs and characterizes their
critical role in regulating neurovascular coupling in the living retina under both
physiological and pathological conditions.
Pericytes embedded along capillary walls regulate microcirculatory
blood flow by contracting and relaxing to induce changes in capil-
lary diameter
4,5
. This model explains capillary dynamics, but does not
account for the need to coordinate dilation and constriction in distal
capillaries to achieve fine regulation of blood supply within a local
network. Examination of retinas from mice thatexpress red fluorescent
protein under control of the NG2 (Cspg4) promoter (NG2–DsRed),
which allows the selective visualization of retinal pericytes6, revealed
fine processes connecting neighbouring capillaries (Fig.1a), remi-
niscent of intervascular bridging strands described in fixed tissue7–13.
The colocalization of DsRed and TRITC-lectin, which labels both the
pericyte’s basement membrane and endothelial cells, showed that
these processes emerged from the pericyte soma and extended to
distal capillaries (Fig.1b, c). These pericyte-derived structures were
tubular (Supplementary Video1), with an average diameter of 500nm
(Fig.1d), resembling thin tunnelling nanotubes
14
. They ranged in length
from 4 to 90μm, with a bimodal length distribution; we classified them
as either short or long (shorter or longer than 30μm), and found that
short processes were more prevalent than long processes (66% and
34%, respectively) (Fig.1e, f). Retinal labelling with phalloidin, which
binds F-actin, revealed that these pericyte-derived processes contain
an F-actin cytoskeleton (Fig.1g, h), a structural requirement of thin
tunnelling nanotubes.
An important phenotypic criterion of thin tunnelling nanotubes
is that they connect two distinct cells14. Analysis of retinas from
NG2–DsRed mice revealed that the pericyte elaborating each thin
tunnelling nanotube, referred to here as the proximal pericyte, con-
nected with the process of a pericyte on a distal capillary, thus termed
the distal pericyte (Extended Data Fig.1a–d). We therefore named
these processes interpericyte tunnelling nanotubes (IP-TNTs). To
investigate whether these structures generally connect two peri-
cytes, we performed single-pericyte electroporation of fluorescein, a
low-molecular-weight dye (332Da). Fluorescein diffused rapidly from
the soma of the proximal pericyte into the IP-TNT and, critically, into
https://doi.org/10.1038/s41586-020-2589-x
Received: 12 December 2018
Accepted: 6 July 2020
Published online: 12 August 2020
Check for updates
1Department of Neuroscience, University of Montreal, Montreal, Quebec, Canada. 2University of Montreal Hospital Research Centre, Montreal, Quebec, Canada. 3Centre for Research in
Neuroscience, Department of Neurology and Neurosurgery, The Research Institute of the McGill University Health Centre, Montreal General Hospital, Montreal, Quebec, Canada. 4These
authors contributed equally: Luis Alarcon-Martinez, Deborah Villafranca-Baughman. e-mail: lalarcon@um.es; adriana.di.polo@umontreal.ca
Content courtesy of Springer Nature, terms of use apply. Rights reserved
... Here, our work confirmed that TNTs are elastic structures that bend under radial force. Indeed, in vivo observations show that curved TNT-like structures present in brain and embryo tissue (Osswald et al., 2015;Alarcon-Martinez et al., 2020;Scheiblich et al., 2021), indicating that they tolerate radial forces derived from adjacent cells and extracellular matrix. Furthermore, we measured the spring constant of TNTs (79.1 ± 16.2 pN/μm). ...
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