Nils Korte: Inhibiting Ca2+ channels in Alzheimer’s disease model mice relaxes pericytes, improves cerebral blood flow and reduces immune cell stalling and hypoxia

This may be because, when therapies are started, irreversible neuronal damage has already occurred, prompting a search for new therapeutic targets that are useful early in the disease. One such target is cerebral blood flow (CBF). CBF is reduced by approximately 45% in affected brain areas in AD3, sufficient to result in loss of attention, myelinated axon disruption, spatial memory deficits and synapse loss4,5,6. CBF decrease is associated with increased capillary transit time heterogeneity, which amplifies tissue hypoxia7. A causal influence of blood flow changes on cognitive changes early in AD, before synapses or neurons are lost, is suggested by the CBF reduction starting early8,9 in preclinical AD, with a faster onset than the deposition of Aβ or tau10, and correlating with cognitive decline7,11.

In human AD, this CBF decrease is associated with capillary constriction by contractile pericytes12. This may reflect Aβ stimulating production of reactive oxygen species (ROS) that release the vasoconstrictor endothelin-1 (ET-1)12. Pericyte-mediated capillary constriction is also seen in the APPNL–G–F mouse model of AD, in which there was no constriction of arterioles or venules, implicating pericytes as causing the CBF decrease12. Pericyte contraction is evoked by a rise of intracellular calcium concentration ([Ca2+]i) or by activating the Rho kinase pathway13,14,15,16. It is best characterized for the 1st–3rd capillary branch orders (from penetrating arterioles (PAs)), for which pericytes have circumferential processes close to their somata12, but even pericytes with less circumferential processes on higher-order capillary branches are now thought to be contractile and to regulate CBF15,17.

The contraction of pericytes decreases CBF in three ways. First, the capillary diameter reduction that it produces decreases the cross-sectional area available for flow, which increases resistance via Poiseuille’s law. Second, it leads to an increase in blood viscosity, by promoting interaction of cells in the blood with the vessel walls18. Finally, blood cells can get stuck in vessels where the diameter is reduced14,19. This is especially important for leukocytes, which are larger and less deformable than red blood cells (RBCs).

Agents reducing pericyte contraction in AD should increase CBF by increasing capillary diameter, decreasing blood viscosity and reducing block of capillaries. They might maintain normal neuronal function for longer and delay irreversible deleterious effects on neurons. In the present study, we examined using the blood–brain barrier (BBB)-permeable voltage-gated calcium channel (CaV) blocker nimodipine for this purpose. Combining measurements of pericyte [Ca2+]i, capillary diameter, blood flow (from laser speckle, laser Doppler and magnetic resonance imaging (MRI)) and capillary stalling, we show that, in an AD mouse model, nimodipine lowers pericyte [Ca2+]i, thus relaxing pericytes throughout the capillary bed, dilating capillaries and reducing capillary block by neutrophils and other cells. Consequently, CBF is increased and tissue hypoxia reduced.

Results

The morphology of mural cells (smooth muscle cells (SMCs) and pericytes) at different positions in the capillary bed is shown in Extended Data Fig. 1a. Experiments were initially performed on pericytes of the 1st, 2nd and 3rd capillary branching order from PAs, where 1st order is the first branch off the PA; 2nd order is a branch off the 1st order, etc.

L-type Ca2+ and TMEM16A Cl− channels control pericyte tone

Pericyte contractile tone is controlled by Ca2+ via an interaction between L-type CaVs and the Ca2+-gated Cl− channel TMEM16A (ref. 14). Our strategy for reducing the CBF decrease that occurs early in AD is to prevent a [Ca2+]i rise in pericytes. Extended Data Fig. 1b–g shows how blockers of CaVs and TMEM16A can achieve this. Gq-protein-coupled receptor (GqPCR) agonists, such as ET-1 (a driver of Aβ-evoked pericyte contraction in AD12), release Ca2+ from sarcoplasmic reticulum. This Ca2+ may activate actomyosin directly or trigger Cl− exit via TMEM16A channels14, leading to membrane depolarization and CaV activation, causing Ca2+ influx and contraction. Two-photon imaging of acute cortical slices, from mice with mural cells expressing tdTomato and the calcium indicator GCaMP5g (NG2-CreERT2-GCaMP5g mice), was performed. ET-1 increased pericyte [Ca2+]i and constricted capillaries at pericyte somata (Extended Data Fig. 1b–d) where most circumferential pericyte processes mediating constriction12 are present (Extended Data Fig. 2a and Supplementary Video 1). Blocking CaVs with nimodipine, or TMEM16A with 10bm (ref. 20), greatly attenuated the [Ca2+]i rise and pericyte contraction (Extended Data Fig. 1b–d), and a similar decrease by nimodipine of the ET-1 evoked [Ca2+]i rise was detected in SMCs (Extended Data Fig. 1e), consistent with CaV1.2 (Extended Data Fig. 3) and TMEM16A (ref. 14) being highly expressed in mural cells. The vehicle used to dissolve nimodipine did not modulate [Ca2+]i in pericytes (Extended Data Fig. 1f). Our model, underpinning this paper, of how TMEM16A and CaVs control pericyte tone is shown in Extended Data Fig. 1g.

CaVs generate myogenic tone in SMCs and pericytes in vivo

Our understanding of pericyte contraction comes mainly from experiments on brain slices. In vivo, intravascular pressure and shear stress release vasoactive messengers from endothelial cells and may cause SMCs and pericytes to constrict vessels. To assess whether CaVs generate contractile tone in vivo, we measured CBF using laser Doppler flowmetry with in vivo two-photon imaging of the cerebral vasculature in urethane-anesthetized NG2-dsRed mice that have pericytes and SMCs labeled with dsRed21 (Fig. 1a,b). FITC-dextran (70 kDa, intravenous (i.v.)) was used to visualize blood flow. Femoral vein infusion of the BBB-permeable CaV blocker nimodipine (220 μg kg−1 total, over 10 min, 60 μg ml−1 solution) evoked a long-lasting increase in CBF of 28% in nine anesthetized mature P120–P158 wild-type (WT) mice (Fig. 1b,c; ‘mature’ is used for mice aged P110–P200). Infusion of vehicle alone did not alter CBF (Fig. 1c), implying that nimodipine, rather than the vehicle or the small increase in blood volume, raises the CBF.

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