Pericytes are perivascular mural cells of brain capillaries that are positioned centrally within the neurovascular unit between endothelial cells, astrocytes and neurons. This unique position allows them to play a major role in regulating key neurovascular functions of the brain. The role of pericytes in the regulation of cerebral blood flow (CBF) and neurovascular coupling remains, however, debatable. Using loss-of-function pericyte-deficient mice, here we show that pericyte degeneration diminishes global and individual capillary CBF responses to neuronal stimulus resulting in neurovascular uncoupling, reduced oxygen supply to brain and metabolic stress. We show that these neurovascular deficits lead over time to impaired neuronal excitability and neurodegenerative changes. Thus, pericyte degeneration as seen in neurological disorders such as Alzheimer’s disease may contribute to neurovascular dysfunction and neurodegeneration associated with human disease.

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Pericytes are positioned between brain capillary endothelial cells, astrocytes and neurons. They degenerate in multiple neurological disorders. However, their role in the pathogenesis of these disorders remains debatable. Here, we generated an inducible pericyte-specific Cre line and crossed pericyte-specific Cre mice with iDTR mice carrying Cre-dependent human diphtheria toxin receptor (DTR). After pericyte ablation with diphtheria toxin, mice developed an acute blood-brain barrier (BBB) breakdown, severe loss of blood flow, and a rapid neuron loss associated with loss of pericyte-derived pleiotrophin (PTN), a neurotrophic growth factor. Intracerebroventricular PTN infusions prevented neuron loss in pericyte-ablated mice despite persistent circulatory changes. Silencing pericyte-derived Ptn rendered neurons vulnerable to ischemic and excitotoxic injury. Our data demonstrate a rapid neurodegeneration cascade linking pericyte loss to acute circulatory collapse and loss of PTN neurotrophic support. These findings could have implications for the pathogenesis and treatment of neurological disorders associated with pericyte loss and/or neurovascular dysfunction.

he APOE4 variant of apolipoprotein E is the strongest genetic risk factor for AD 1 . One and two APOE4 alleles increase risk for AD by approximately 4-and 15-fold, respectively, compared to the more-common APOE3 gene that carries lower risk for AD 1 . Besides accelerating onset and progression of dementia, APOE4 is associated with different brain pathologies. For example, APOE4 accelerates BBB breakdown and degeneration of brain capillary pericytes 2,3 that maintain BBB integrity [4][5][6] and leads to cerebral blood flow (CBF) reduction 7,8 and dysregulation 7,9,10 . APOE4 is toxic to neurons 11 and accelerates tau-mediated neurodegeneration 12 . Additionally, APOE4 slows down amyloid-β (Aβ) clearance 13,14 and accelerates amyloid deposition [14][15][16] , which promotes development of amyloid pathology.Recent studies focused on very early stages in the Alzheimer's continuum in individuals who are cognitively unimpaired or with mild cognitive impairment (MCI) have shown that individuals bearing an APOE4 variant (APOE3/APOE4 or APOE4/APOE4) are distinguished from APOE3 homozygotes by breakdown in the BBB in the hippocampus and medial temporal lobe, regions responsible for memory encoding and cognitive functions 17 . This finding is apparent in cognitively unimpaired APOE4 carriers and more severe in those with MCI and is independent of Aβ or tau pathology measured in the cerebrospinal fluid or in brain by positron emission tomography 17 . These findings support the growing evidence suggesting that vascular dysfunction, BBB breakdown and vascular disorder contribute to early cognitive impairment and AD [17][18][19][20][21][22][23][24][25][26] . On the other hand, accumulation of Aβ in the brain has also been suggested to occur years before cognitive impairment and continues to increase with disease progression 27 . Although it has been shown that vascular dysfunction contributes to early cognitive impairment in ways that may not be exclusively related to classical AD pathology 17,19,20,26 , the respective contributions of the BBB pathway and vascular disorder versus amyloid-β pathway to advanced disease stage during progression of neurodegenerative disorder and cognitive decline in AD are still poorly understood.To address this question, here we studied vascular dysfunction, Aβ pathology, neuronal dysfunction and behavior in older APOE3 and APOE4 knock-in mice 28 alone and crossed with the 5xFAD line 29 . All mice were derived from the same litters, as previously described 30 . Mice lacking Apoe 3 and/or expressing human APOE4 develop early BBB breakdown 3,[31][32][33] and CBF dysregulation 10 . On the other hand, the 5xFAD line also develops BBB breakdown [34][35][36][37] , CBF reductions 38 and neuron and synaptic loss at a later stage 29,39 , whereas APOE3;5xFAD and APOE4;5xFAD mice (also known as E3FAD and E4FAD lines, respectively 30 ) have comparable Aβ pathology at an older age 40 . These features of the studied models allowed us to interrogate how different pathologies in APOE4 compared to APOE3 mice relat...

3K3A-activated protein C (APC), a cell-signaling analogue of endogenous blood serine protease APC, exerts vasculoprotective, neuroprotective, and anti-inflammatory activities in rodent models of stroke, brain injury, and neurodegenerative disorders. 3K3A-APC is currently in development as a neuroprotectant in patients with ischemic stroke. Here, we report that 3K3A-APC inhibits BACE1 amyloidogenic pathway in a mouse model of Alzheimer’s disease (AD). We show that a 4-mo daily treatment of 3-mo-old 5XFAD mice with murine recombinant 3K3A-APC (100 µg/kg/d i.p.) prevents development of parenchymal and cerebrovascular amyloid-β (Aβ) deposits by 40–50%, which is mediated through NFκB–dependent transcriptional inhibition of BACE1, resulting in blockade of Aβ generation in neurons overexpressing human Aβ-precursor protein. Consistent with reduced Aβ deposition, 3K3A-APC normalized hippocampus-dependent behavioral deficits and cerebral blood flow responses, improved cerebrovascular integrity, and diminished neuroinflammatory responses. Our data suggest that 3K3A-APC holds potential as an effective anti-Aβ prevention therapy for early-stage AD.

Cerebrovascular dysfunction has an important role in the pathogenesis of multiple brain disorders. Measurement of hemodynamic responses in vivo can be challenging, particularly as techniques are often not described in sufficient detail and vary between laboratories. We present a set of standardized in vivo protocols that describe high-resolution two-photon microscopy and intrinsic optical signal (IOS) imaging to evaluate capillary and arteriolar responses to a stimulus, regional hemodynamic responses, and oxygen delivery to the brain. The protocol also describes how to measure intrinsic NADH fluorescence to understand how blood O supply meets the metabolic demands of activated brain tissue, and to perform resting-state absolute oxygen partial pressure (pO) measurements of brain tissue. These methods can detect cerebrovascular changes at far higher resolution than MRI techniques, although the optical nature of these techniques limits their achievable imaging depths. Each individual procedure requires 1-2 h to complete, with two to three procedures typically performed per animal at a time. These protocols are broadly applicable in studies of cerebrovascular function in healthy and diseased brain in any of the existing mouse models of neurological and vascular disorders. All these procedures can be accomplished by a competent graduate student or experienced technician, except the two-photon measurement of absolute pO level, which is better suited to a more experienced, postdoctoral-level researcher.

Pericytes are perivascular mural cells that enwrap brain capillaries and maintain bloodbrain barrier (BBB) integrity. Most studies suggest that pericytes regulate cerebral blood flow (CBF) and oxygen delivery to activated brain structures, known as neurovascular coupling. While we have previously shown that congenital loss of pericytes leads over time to aberrant hemodynamic responses, the effects of acute global pericyte loss on neurovascular coupling have not been studied. To address this, we used our recently reported inducible pericyte-specific Cre mouse line crossed to iDTR mice carrying Cre-dependent human diphtheria toxin (DT) receptor, which upon DT treatment leads to acute pericyte ablation. As expected, DT led to rapid progressive loss of pericyte coverage of cortical capillaries up to 50% at 3 days post-DT, which correlated with approximately 50% reductions in stimulus-induced CBF responses measured with laser doppler flowmetry (LDF) and/or intrinsic optical signal (IOS) imaging. Endothelial response to acetylcholine, microvascular density, and neuronal evoked membrane potential responses remained, however, unchanged, as well as arteriolar smooth muscle cell (SMC) coverage and functional responses to adenosine, as we previously reported. Together, these data suggest that neurovascular uncoupling in this model is driven by pericyte loss, but not other vascular deficits or neuronal dysfunction. These results further support the role of pericytes in CBF regulation and may have implications for neurological conditions associated with rapid pericyte loss such as hypoperfusion and stroke, as well as conditions where the exact time course of global regional pericyte loss is less clear, such as Alzheimer's disease (AD) and other neurogenerative disorders.

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