Potassium channels, Neurovascular Coupling, CADASIL

Mount Mansfield

University of Vermont

Burlington, Vermont

September 20, 2013 Second International Workshop on CADASIL Mark Nelson

"And what physicians say about disease is applicable here: that at the beginning a disease is easy to cure but difficult to diagnose; but as time passes, not having been treated or recognized at the outset, it becomes easy to diagnose but difficult to cure. The same thing occurs in affairs of state; for by recognizing from afar the diseases that are spreading in the state (which is a gift given only to a prudent ruler), they can be cured quickly; but when they are not recognized and are left to grow to the extent that everyone recognizes them, there is no longer any cure. Niccolo Machiavelli (aka Fabrice Dabertrand)

Pathogenesis of Small Vessel Disease of the Brain

Fondation Leducq Transatlantic Network of Excellence

North American Coordinator Mark T. NELSON (USA) University Distinguished Professor and Chair Department of Pharmacology College of Medicine University of Vermont Burlington, USA Mark.Nelson@uvm.edu Phone: 802 656 2500, Fax: 802 656 4523

European Coordinator Anne JOUTEL (France) INSERM Research Director Genetics of Vascular Diseases LaboratoryINSERM University Paris7-Denis Diderot 10 avenue de Verdun, 75010 Paris, France anne.joutel@univ-paris-diderot.fr Phone: 01 5727 8589, Fax: 01 5727 8594

Brain survival
Autoregulation: Maintaining constant blood flow in response to blood pressure fluctuations. Neurovascular coupling: Regional coupling of cerebral blood flow to the metabolic demand of neurons.
The brain possesses intrinsic mechanisms by which its vascular supply can be varied locally in correspondence with local variations of functional activity (Roy and Sherrington, 1890)

Implications of neurovascular coupling

Functional brain imaging by monitoring local cerebral blood flow with fMRI. Understanding of brain diseases that are associated with neurovascular dysfunction, including stroke, Alzheimers, vascular dementia, hypertension. Modulation of neural processing.

The Neurovascular Unit Arterioles in the brain are encased by astrocytic processes

Iadecola, Nature Reviews, Neuroscience, 2004

The Neurovascular Unit

Neurons Astrocyte endfeet

Pericyte

Endothelium

10 m

Smooth muscle

Astrocyte endfeet completely encase the intracerebral vasculature and are thus uniquely positioned to deliver signaling molecular to the underlying smooth muscle Endothelial cells line the lumen of the arterioles, and communicate blood-borne signals to smooth muscle to affect tone

Neurovascular coupling

A plastic emulsion was injected into the brain vessels, and brain parenchymal tissue was dissolved (Zlokovic & Apuzzo, 1998; Lippincott Williams & Wilkins)

Level of astrocytic endfoot Ca2+ determines whether an adjacent arteriole dilates or constricts depending on perivascular K+.

Dunn, K. M. and M. T. Nelson (2010). "Potassium Channels and Neurovascular Coupling." Circulation Journal 74(4): 608-616

CADASIL

Cerebral
Autosomal

Inherited small vessel disease causing stroke and subcortical vascular dementia that starts in early adulthood and progresses over time. Caused by mutations in NOTCH3 gene

Dominant
Arteriopathy with

Subcortical
Infarcts &

EGFR10 and 11 required for ligand biding

Leucoencephalopathy

Mechanisms linking Notch3 mutation to vascular impairment remain enigmatic

From Chabriat H et al. Lancet Neurol. 2009 Jul;8(7):643-53. Review.

The mouse model

Loss of Notch3 causes structural and functional alterations of small arteries, but does not cause CADASIL pathology. (Liu et al Circ Res 2010)
Anne Joutel and coworkers used a P1-derived artificial chromosome-based transgenesis to overexpress Notch3 in an endogenous-like expression pattern. Line 129 overexpresses (x4) rat wild-type Notch3 gene, and line 88 overexpresses (x4) a CADASIL form Notch3R169C .

Non-Tg

TgNotch3WT

TgNotch3R169C

Notch3 Notch3 Notch3 Notch3 Notch3 Notch3 Notch3

Notch3 Notch3 Notch3

Notch3R169C Notch3R169C Notch3R169C Notch3R169C

Notch3R169C Notch3R169C Notch3R169C

Notch3

Notch3 Notch3

Notch3

Notch3R169C

Cerebrovascular dysfunction and microcirculation rarefaction precede white matter lesions

Joutel A et al. J Clin Invest. (2010) 120(2):433-45.

Young TgR169C (5-6 months) mice exhibit altered cerebrovascular autoregulation, attenuated functional hyperemia and a decreased myogenic response in the arteries of the surface of the brain

Whisker stimulation

Myogenic response of posterior cerebral artery

Joutel A et al. J Clin Invest. (2010) 120(2):433-45.

Parenchymal arteriole with parent middle cerebral artery (Red, aquaporin 4 (astrocyte) staining) (From Hannah et al., JCBFM, 2011)

Roi Ca2+ movies only

Membrane potential and diameter measurements of pressurized parenchymal arteriole

Monitor Tie Edge detection system Cannula

Pressurized PA (40 mm Hg)

microelectrode Membrane potential (mV)

CADASIL parenchymal arterioles constrict less to intravascular pressure

Upregulation of potassium channel activity to oppose increases in intravascular pressure

one way ANOVA

Resistance-sized mesenteric arteries display normal myogenic tone

Ion channel control of smooth muscle membrane potential and intracellular calcium

Kv: Voltage-dependent potassium channel. BKCa: Large-conductance, calcium-sensitive potassium channel. Kir: Strong inward rectifier potassium channel. Activated by external K+ and membrane potential hyperpolarization. IKCa: Intermediate conductance, calcium-activated potassium channel. SKCa: Small conductance, calcium-activated potassium channel.

External K+o is a potent and rapid vasodilator and constrictor of pressurized (80 mm Hg) cerebral arteries
External [K ] (mM)
+

Nisoldipine (10 nM)

61 81

Calcium channel inhibitor

Arterial Wall [Calcium], nM

51 31 36 41 26 21 6 11 16

350 300 250 200 150 100 50 20 40 80 100

Astrocytic Endfoot Calcium

BK Perivascular K+

Time, min

Arterial Diameter, m

Dilation to Kir2.1 activation

200 150 100 50 0

Dilation Constriction <20 mM >20 mM

20 40 80 100

Knot et al., J. Physiol., 1998

Time, min

Membrane potential of vascular smooth muscle determines arterial diameter and hence blood flow (External K+ regulates membrane potential)
Calcium channels inhibited Death

Life

Death

Knot & Nelson, J Physiol 1998

Dilation and constriction to elevation of [K+]0 from 3 to 60 mM are unaffected in CADASIL

Membrane potential of arteriolar smooth muscle from CADASIL mice is hyperpolarized at 40 mm Hg

Vm (mV)
Non-Tg -52.3 0.8 -52.05 1.1 -53.6 2.1 10 mm Hg -34 0.6 -35.7 1.6 -44.2 1.4 40 mm Hg

TgControl

TgR169C

one way ANOVA

Ion channel control of smooth muscle membrane potential and intracellular calcium

Kv: Voltage-dependent potassium channel. BKCa: Large-conductance, calcium-sensitive potassium channel. Kir: Strong inward rectifier potassium channel. Activated by external K+ and membrane potential hyperpolarization. IKCa: Intermediate conductance, calcium-activated potassium channel. SKCa: Small conductance, calcium-activated potassium channel.

Direct measurements of potassium channel activity using the patch clamp technique
+60 mV

-40 mV -80 mV -70 mV

Objective: compare currents from isolated arteriolar smooth muscle cell elicited by voltage pulses from -70 mV to +60 mV. Experiment conducted in the absence of Ca2+, and in presence of BK blocker paxilline

Amphotericin B pore

Adapted from http://patch-clamp.info/techniques/techniques.htm

Current density of voltage-dependent K+ channels is greater in CADASIL (cell membrane surface area is unaffected)

Current densities are different from -30 mV to 60 mV

one way ANOVA

Cerebral Blood Flow autoregulation is altered in CADASIL

Pressure-induced constrictions are unaltered at lower pressures (<30 mm Hg) but myogenic tone is decreased at physiological pressures in both pial arteries and parenchymal arterioles (PAs) from CADASIL mice. Implication: Pressure-dependent mechanism is altered in CADASIL. However, contractile function is preserved with diameter changes to potassium being unaltered. PAs from CADASIL mice show a more hyperpolarized membrane potential at 40 mm Hg. BK, IK, SK channels and eNOS function do not appear different in CADASIL mouse. Voltage-dependent potassium (Kv) channel current density is elevated in cerebrovascular smooth muscle cells from CADASIL mice.

BK, EC function (IK, SK, eNOS)

Pressure

Vm depolarization

[Ca2+]i Kv

Diameter

CBF

Subarachnoid hemorrhage blood increases pressure-induced constrictions through downregulation of voltage-dependent potassium channels

Masayo Koide Adrian Bonev George Wellman

Kv channel trafficking is regulated by metalloproteinase (MMP) through heparin binding epidermal growth factor like growth factor (EGFR)

Koide et al. AJP 2007 Ishiguro et al. Circ Res 2006

Heparin-binding Epidermal Growth Factor (HB-EGF) induces Kv current suppression

Koide et al. AJP Heart Circ Physiol. 2007 Sep;293(3):H1750-9

HB-EGF restores myogenic response in cerebral arteries from CADASIL mouse

Paired T-test

Excess of Notch3ECD abnormally stabilizes TIMP3 (GOM formation), which increases Kv channels at the membrane to oppose pressure-induced constriction
shedding

GOM formation TIMP3

Notch3ECD

Notch3ICD

Gene expression (Kv 1.5?) From Koide 2007

Two approaches to restore myogenic tone in CADASIL

Pharmacologic activation of EGFR restores myogenic tone in parenchymal arterioles (in Burlington)

Genetic reduction of TIMP-3 restores myogenic tone in posterior cerebral arteries (in Paris)

A mechanism by which Notch3 mutation leads to a vascular impairment

Pleiotropic effect of Notch3R169C via GOM?

Neuronal activity AA Prostaglandins/ prostacyclin PLA2 Ca2+ wave

Astrocyte endfoot

BK

GOM formation
20-HETE

K+

K+

K+

K+ Kir

Dilation

EET

Constriction

EM

+
Local blood flow

Dilation Endothelium

Local blood flow

Cartoon from Gro Klitgaard Povlsen

Smooth muscle

Granular Osmiophilic Material (GOM) are detected between smooth muscle cells and astrocytic endfoot

1 m
From Anne Joutel

METHODS
1. Coronal brain slices from neonatal rats P5-P27

Adapted from Methods for Slice Preparation and General Use (Tensor Biosciences) by Daniel H. Chun

Vascular tone induced with the thromboxane agonist U46619 (125 nM )

Two-photon

Brain slices loaded with Fluo-4.AM

DIC-IR+fluorescence
EFS, Ca2+ uncaging

.
20X

Neurovascular coupling: Elevation of astrocytic endfoot Ca2+ causes vasodilation

EFS = Electrical Field Stimulation

EFS-induced dilation is reduced in CADASIL mice

EFS-induced Ca2+ increase in astrocytic endfoot is reduced in CADASIL mice

Faster [Ca2+] decay in endfeet from CADASIL (TgR169C) mice

K Half Life Tau 0.05497 0.1321 12.61 5.246 18.19 7.568

TgWT

TgR169C

Working Hypothesis: Early consequence of CADASIL is upregulation of Kv 1.5 expression and activity which leads to compromised autoregulation. Elevation of the metalloproteinase TIMP3 in GOM leads to an increase in surface expression of Kv channels. Decreased myogenic tone would diminish vasodilator capacity, and thereby neurovascular coupling. CADASIL likely attenuates astrocyte endfoot calcium signaling to decease neurovascular coupling.

Acknowledgements

Funding: American Heart Association, Totman Medical Research Trust, Lundbeck Foundation National Institute of Health,Fondation Leducq

Adrian Bonev
Jonathan Ledoux Fabrice Dabertrand Christel Krigaard

Anne Joutel

Carmen Capone

Merci! Danke! Thank You!

The Lab