Atorvastatin rescues hyperhomocysteinemia-induced cognitive deficits and neuroinflammatory gene changes.

BACKGROUND: Epidemiological data suggests statins could reduce the risk of dementia, and more specifically, Alzheimer's disease (AD). Pre-clinical data suggests statins reduce the risk of dementia through their pleiotropic effects rather than their cholesterol lowering effects. While AD is a leading cause of dementia, it is frequently found co-morbidly with cerebral small vessel disease and other vascular contributions to cognitive impairment and dementia (VCID), which are another leading cause of dementia. In this study, we determined if atorvastatin ameliorated hyperhomocysteinemia (HHcy)-induced VCID. METHODS: Wild-type (C57Bl6/J) mice were placed on a diet to induce HHcy or a control diet each with or without atorvastatin for 14 weeks. Mice underwent novel object recognition testing before tissue collection. Plasma total cholesterol and total homocysteine as well as related metabolites were measured. Using qPCR and NanoString technology, we profiled glial cell-associated gene expression changes. Finally, microglial morphology, astrocyte end feet, and microhemorrhages were analyzed using histological methods. RESULTS: Atorvastatin treatment of HHcy in mice led to no changes in total cholesterol but decreases in total homocysteine in plasma. While HHcy decreased expression of many glial genes, atorvastatin rescued these gene changes, which mostly occurred in oligodendrocytes and microglia. Microglia in HHcy mice with atorvastatin were trending towards fewer processes compared to control with atorvastatin, but there were no atorvastatin effects on astrocyte end feet. While atorvastatin treatment was trending towards increasing the area of microhemorrhages in HHcy mice in the frontal cortex, it only slightly (non-significantly) reduced the number of microhemorrhages. Finally, atorvastatin treatment in HHcy mice led to improved cognition on the novel object recognition task. CONCLUSIONS: These data suggest that atorvastatin rescued cognitive changes induced by HHcy most likely through lowering plasma total homocysteine and rescuing gene expression changes rather than impacts on vascular integrity or microglial changes.

CU06-1004 (endothelial dysfunction blocker) ameliorates astrocyte end-feet swelling by stabilizing endothelial cell junctions in cerebral ischemia/reperfusion injury.

Cerebral ischemia, or stroke, is widespread leading cause of death and disability. Surgical and pharmacological interventions that recover blood flow are the most effective treatment strategies for stroke patients. However, restoring the blood supply is accompanied by severe reperfusion injury, with edema and astrocyte end-feet disruption. Here, we report that the oral administration of CU06-1004 (previously Sac-1004), immediately after onset of ischemia/reperfusion (I/R), ameliorated cerebral damage. CU06-1004 stabilized blood­brain barrier by inhibiting the disruption of the tight junction-related protein zona occludens-1 and the cortical actin ring in endothelial cells (ECs) after I/R. Interestingly, CU06-1004 significantly suppressed astrocyte end-feet swelling following I/R, by reducing aquaporin 4 and connexin 43 levels, which mediates swelling. Furthermore, the degradation of ß1-integrin and ß-dystroglycan, which anchors to the cortical actin ring in ECs, was inhibited by CU06-1004 administration after I/R. Consistently, CU06-1004 administration following I/R also suppressed the loss of laminin and collagen type IV, which bind to the cortical actin ring anchoring proteins. Unlike the protective effects of CU06-1004 in ECs, astrocyte viability and proliferation were not directly affected. Taken together, our observations suggest that CU06-1004 inhibits I/R-induced cerebral edema and astrocyte end-feet swelling by maintaining EC junction stability. KEY MESSAGES: • CU06-1004 ameliorates I/R-induced cerebral injury. • EC junction integrity was stabilized by CU06-1004 treatment after I/R. • CU06-1004 reduces astrocyte end-feet swelling following I/R. • EC junction stability affects astrocyte end-feet structure maintenance after I/R.

Fast Ca2+ responses in astrocyte end-feet and neurovascular coupling in mice.

Cerebral blood flow (CBF) is regulated by the activity of neurons and astrocytes. Understanding how these cells control activity-dependent increases in CBF is crucial to interpreting functional neuroimaging signals. The relative importance of neurons and astrocytes is debated, as are the functional implications of fast Ca2+ changes in astrocytes versus neurons. Here, we used two-photon microscopy to assess Ca2+ changes in neuropil, astrocyte processes, and astrocyte end-feet in response to whisker pad stimulation in mice. We also developed a pixel-based analysis to improve the detection of rapid Ca2+ signals in the subcellular compartments of astrocytes. Fast Ca2+ responses were observed using both chemical and genetically encoded Ca2+ indicators in astrocyte end-feet prior to dilation of arterioles and capillaries. A low dose of the NMDA receptor antagonist (5R,10s)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine-hydrogen-maleate (MK801) attenuated fast Ca2+ responses in the neuropil and astrocyte processes, but not in astrocyte end-feet, and the evoked CBF response was preserved. In addition, a low dose of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), an agonist for the extrasynaptic GABAA receptor (GABAA R), increased CBF responses and the fast Ca2+ response in astrocyte end-feet but did not affect Ca2+ responses in astrocyte processes and neuropil. These results suggest that fast Ca2+ increases in the neuropil and astrocyte processes are not necessary for an evoked CBF response. In contrast, as local fast Ca2+ responses in astrocyte end-feet are unaffected by MK801 but increase via GABAA R-dependent mechanisms that also increased CBF responses, we hypothesize that the fast Ca2+ increases in end-feet adjust CBF during synaptic activity.

Selective knockout of astrocytic Na+ /H+ exchanger isoform 1 reduces astrogliosis, BBB damage, infarction, and improves neurological function after ischemic stroke.

Stimulation of Na+ /H+ exchanger isoform 1 (NHE1) in astrocytes causes ionic dysregulation under ischemic conditions. In this study, we created a Nhe1flox/flox (Nhe1f/f ) mouse line with exon 5 of Nhe1 flanked with two loxP sites and selective ablation of Nhe1 in astrocytes was achieved by crossing Nhe1f/f mice with Gfap-CreERT2 Cre-recombinase mice. Gfap-CreERT2+/- ;Nhe1f/f mice at postnatal day 60-90 were treated with either corn oil or tamoxifen (Tam, 75 mg/kg/day, i.p.) for 5 days. After 30 days post-injection, mice underwent transient middle cerebral artery occlusion (tMCAO) to induce ischemic stroke. Compared with the oil-vehicle group (control), Tam-treated Gfap-CreERT2+/- ;Nhe1f/f (Nhe1 KO) mice developed significantly smaller ischemic infarction, less edema, and less neurological function deficits at 1-5 days after tMCAO. Immunocytochemical analysis revealed less astrocytic proliferation, less cellular hypertrophy, and less peri-lesion gliosis in Nhe1 KO mouse brains. Selective deletion of Nhe1 in astrocytes also reduced cerebral microvessel damage and blood-brain barrier (BBB) injury in ischemic brains. The BBB microvessels of the control brains show swollen endothelial cells, opened tight junctions, increased expression of proinflammatory protease MMP-9, and significant loss of tight junction protein occludin. In contrast, the Nhe1 KO mice exhibited reduced BBB breakdown and normal tight junction structure, with increased expression of occludin and reduced MMP-9. Most importantly, deletion of astrocytic Nhe1 gene significantly increased regional cerebral blood flow in the ischemic hemisphere at 24 hr post-MCAO. Taken together, our study provides the first line of evidence for a causative role of astrocytic NHE1 protein in reactive astrogliosis and ischemic neurovascular damage.

The blood-brain barrier after stroke: Structural studies and the role of transcytotic vesicles.

Blood-brain barrier breakdown worsens ischaemic damage, but it is unclear how molecules breach the blood-brain barrier in vivo. Using the obese ob/ob mouse as a model of enhanced blood-brain barrier breakdown, we investigated how stroke-induced structural changes to the microvasculature related to blood-brain barrier permeability. Ob/ob mice underwent middle cerebral artery occlusion, followed by 4 or 24 h reperfusion. Blood-brain barrier integrity was assessed using IgG and horseradish peroxidase staining, and blood-brain barrier structure by two-dimensional and three-dimensional electron microscopy. At 4 and 24 h post-stroke, ob/ob mice had increased ischaemic damage and blood-brain barrier breakdown compared to ob/- controls, and vessels from both genotypes showed astrocyte end-foot swelling and increased endothelial vesicles. Ob/ob mice had significantly more endothelial vesicles at 4 h in the striatum, where blood-brain barrier breakdown was most severe. Both stroke and genotype had no effect on tight junction structure visualised by electron microscopy, or protein expression in isolated microvessels. Astrocyte swelling severity did not correlate with tissue outcome, being unaffected by genotype or reperfusion times. However, the rare instances of vessel lumen collapse were always associated with severe astrocyte swelling in two-dimensional and three-dimensional electron microscopy. Endothelial vesicles were therefore the best spatial and temporal indicators of blood-brain barrier breakdown after cerebral ischaemia.

Store-Operated Calcium Entry in Müller Glia Is Controlled by Synergistic Activation of TRPC and Orai Channels.

The endoplasmic reticulum (ER) is at the epicenter of astrocyte Ca(2+) signaling. We sought to identify the molecular mechanism underlying store-operated calcium entry that replenishes ER stores in mouse Müller cells. Store depletion, induced through blockade of sequestration transporters in Ca(2+)-free saline, induced synergistic activation of canonical transient receptor potential 1 (TRPC1) and Orai channels. Store-operated TRPC1 channels were identified by their electrophysiological properties, pharmacological blockers, and ablation of the Trpc1 gene. Ca(2+) release-activated currents (ICRAC) were identified by ion permeability, voltage dependence, and sensitivity to selective Orai antagonists Synta66 and GSK7975A. Depletion-evoked calcium influx was initiated at the Müller end-foot and apical process, triggering centrifugal propagation of Ca(2+) waves into the cell body. EM analysis of the end-foot compartment showed high-density ER cisternae that shadow retinal ganglion cell (RGC) somata and axons, protoplasmic astrocytes, vascular endothelial cells, and ER-mitochondrial contacts at the vitreal surface of the end-foot. The mouse retina expresses transcripts encoding both Stim and all known Orai genes; Müller glia predominantly express stromal interacting molecule 1 (STIM1), whereas STIM2 is mainly confined to the outer plexiform and RGC layers. Elimination of TRPC1 facilitated Müller gliosis induced by the elevation of intraocular pressure, suggesting that TRPC channels might play a neuroprotective role during mechanical stress. By characterizing the properties of store-operated signaling pathways in Müller cells, these studies expand the current knowledge about the functional roles these cells play in retinal physiology and pathology while also providing further evidence for the complexity of calcium signaling mechanisms in CNS astroglia. SIGNIFICANCE STATEMENT: Store-operated Ca(2+) signaling represents a major signaling pathway and source of cytosolic Ca(2+) in astrocytes. Here, we show that the store-operated response in Müller cells, radial glia that perform key structural, signaling, osmoregulatory, and mechanosensory functions within the retina, is mediated through synergistic activation of transient receptor potential and Orai channels. The end-foot disproportionately expresses the depletion sensor stromal interacting molecule 1, which contains an extraordinarily high density of endoplasmic reticulum cisternae that shadow neuronal, astrocytic, vascular, and axonal structures; interface with mitochondria; but also originate store-operated Ca(2+) entry-induced transcellular Ca(2+) waves that propagate glial excitation into the proximal retina. These results identify a molecular mechanism that underlies complex interactions between the plasma membrane and calcium stores, and contributes to astroglial function, regulation, and response to mechanical stress.