PubMed: 30046111

Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease.
Da Mesquita S | Kipnis J | Acton ST | Bai R | Baker W | Cao R | Contarino C | Cornelison RC | Dabhi N | Farber E | Hu S | Kingsmore KM | Lopes MB | Louveau A | Munson JM | Onengut-Gumuscu S | Overall CC | Powell RD | Raper D | Rich SS | Smirnov I | Vaccari A | Viar KE

Evidence a73ba1180c

By contrast, the parenchyma of the CNS is devoid of lymphatic vasculature2; in the brain, removal of cellular debris and toxic molecules, such as amyloid-β peptides, is mediated by a combination of transcellular transport mechanisms across the blood−brain and blood−cerebrospinal fluid (CSF) barriers7–9, phagocytosis and digestion by resident microglia and recruited monocytes and/or macrophages10,11, as well as CSF influx and ISF efflux through a paravascular (glymphatic) route12–14

Evidence 01800d6961

Analysis of lymphoid and myeloid cell populations in the meninges (Extended Data Fig. 9d) demonstrated a significant increase in the number of macrophages upon lymphatic ablation compared to both control groups (Extended Data Fig. 9e), which might be correlated with increased amyloid-β deposition and inflammation in the meninges

Evidence 8e463be75d

Staining for amyloid-β in the brains of nine patients with Alzheimer’s disease and eight controls without Alzheimer’s disease (Extended Data Table 1) revealed, as expected, marked parenchymal deposition of amyloid-β in the brains of patients with Alzheimer’s disease, but not in the brains of the controls without Alzheimer’s disease (Extended Data Fig. 9l, m)

Evidence 5fd5307354

Notably, when compared to tissue from controls, all samples from patients with Alzheimer’s disease demonstrated striking vascular amyloid-β pathology in the cortical leptomeninges (Extended Data Fig. 9l, m) and amyloid-β deposition in the dura mater adjacent to the superior sagittal sinus (Fig. 3i, j) or further away from the sinus (Fig. 3k, l)

Evidence f4ba5d7d2e

These findings showed that prominent meningeal amyloid-β deposition observed in patients with Alzheimer’s disease is also observed in mouse models of Alzheimer’s disease after meningeal lymphatic vessel ablation

Evidence 1ec417cb2a

Macrophages in the dura of cases with Alzheimer’s disease were also found in close proximity to amyloid-β deposits (Fig. 3l)

Evidence def6c8b8dd

Together, three different models of impaired meningeal lymphatic function (pharmacological, surgical and genetic) showed a significant impact on brain perfusion by CSF macromolecules

Evidence c0e5e8cf04

The use of this method resulted in effective ablation of meningeal lymphatic vessels (Fig. 1b, c), without any detectable off-target effects in the coverage of meningeal blood vasculature seven days after the procedure (Fig. 1d)

Evidence 45a3cf25f6

A significant reduction in OVA-A647 drainage was observed in the visudyne with photoconversion group compared to the control groups (Extended Data Fig. 1b)

Evidence be88e15647

Brain perfusion by the CSF tracer was found to be significantly lower in the visudyne with photoconversion group than in the control groups (Fig. 1e, f and Extended Data Fig. 2f, g)

Evidence 7e227e770a

Notably, along with the lower influx of Gd into the parenchyma, we observed higher contrast in signal intensity (over approximately 52 min) in the ventricles of visudyne-treated mice, suggesting that Gd accumulation in the CSF occurred (Extended Data Fig. 3n)

Evidence 6c9cb43ed1

Extracellular deposition of amyloid-β aggregates, the main constituent of senile plaques, is considered to be a pathological hallmark of Alzheimer’s disease that contributes to neuronal dysfunction and behavioural changes

Evidence 7466e0fc99

The ageing-associated decrease in paravascular recirculation of CSF and ISF is thought to be responsible, at least in part, for the accumulation of amyloid-β in the brain parenchyma

Evidence 80a5e9d311

Here we show that meningeal lymphatic vessels have an essential role in maintaining brain homeostasis by draining macromolecules from the CNS (both CSF and ISF) into the cervical lymph nodes

Evidence fccb0c3a21

These findings, as has been suggested previously, demonstrate that the efflux of parenchymal and/or ISF macromolecules and the drainage of these macromolecules into dCLNs are impaired as a consequence of meningeal lymphatic ablation, thus functionally connecting meningeal lymphatics with CSF influx and ISF efflux mechanisms

Evidence 60a633ca98

Notably, the rate of tracer influx into the brain parenchyma was significantly increased as a result of enhanced meningeal lymphatic function (Fig. 2k, l and Extended Data Fig. 6q, r)

Evidence f5f9645638

Similar findings for brain perfusion by CSF were observed when meningeal lymphatic drainage was disrupted by surgical ligation of the vessels afferent to the dCLNs (Extended Data Fig. 3a–d)

Evidence 837c5b3b99

Prospero homeobox protein 1 heterozygous (Prox1+/−) mice, a genetic model of lymphatic vessel malfunction25, also presented impaired perfusion through the brain parenchyma and impaired CSF drainage (Extended Data Fig. 3e–i)

Evidence 40ff67d0c8

The increased drainage after VEGF-C treatment in old mice also correlated with enhanced brain perfusion by CSF macromolecules (Extended Data Fig. 7f, g)

Evidence bf1db5da20

A significant difference between control groups and visudyne with photoconversion group was observed in the cued test of the CFC (Extended Data Fig. 5e, f), which points to an impairment in fear memory and in hippocampal– amygdala neuronal circuitry in mice with impaired meningeal lymphatic vessel function

Evidence ed81de2cd2

Mice with ablated meningeal lymphatic vessels also showed significant deficits in spatial learning in the MWM (Fig. 1k–o)

Evidence bcc8eb778e

Similar impairments in spatial learning and memory were observed in mice that had undergone lymphatic ligation (Extended Data Fig. 5g–j), supporting the notion that the observed effect is a result of dysfunctional meningeal lymphatic drainage and not an artefact of the ablation method using visudyne

Evidence c99d2c9719

Significant transcriptional alterations were also associated with excitatory synaptic remodelling and plasticity, hippocampal neuronal transmission, learning and memory and ageing-related cognitive decline (Extended Data Fig. 5q, r)

Evidence c78f3ffa8e

However, significant differences in hippocampal gene expression were found in response to MWM performance after prolonged meningeal lymphatic ablation (Extended Data Fig. 5m, n)

Evidence 9fcbd9a6df

Notably, although the fold change in significantly altered genes after lymphatic ablation and MWM was moderate (−1.79 < log2(fold change) < 1.69), functional enrichment analysis (Extended Data Fig. 5o, p) revealed changes in gene sets associated with neurodegenerative diseases, such as Huntington’s, Parkinson’s and Alzheimer’s disease (Extended Data Fig. 5o)

Evidence 7b4fa544be

Furthermore, different gene sets that are involved in the regulation of metabolite generation and processing, glycolysis and mitochondrial respiration and oxidative stress were also significantly altered in the hippocampus upon lymphatic ablation and performance of the behaviour test (Extended Data Fig. 5p, s–v)

Evidence 5e741c4264

The reported findings that ageing is also associated with peripheral lymphatic dysfunction led us to hypothesize that the deterioration of meningeal lymphatic vessels underlies some aspects of age-associated cognitive decline

Evidence dd06e089a0

Collectively, these data point to no apparent meningeal lymphatic dysfunction in transgenic mice with Alzheimer’s disease at younger ages, which might explain the inefficacy of mVEGF-C treatment

Evidence 0a53cc86b3

However, 5xFAD mice with ablated meningeal lymphatic vessels demonstrated marked deposition of amyloid-β in the meninges (Fig. 3b), as well as macrophage recruitment to large amyloid-β aggregates (Fig. 3c)

Evidence 4db20abf03

Notably, along with meningeal amyloid-β pathology, we observed an aggravation of brain amyloid-β burden in the hippocampi of 5xFAD mice with dysfunctional meningeal lymphatic vessels (Fig. 3d–g)

Evidence 9d4507679f

A similar outcome was observed in J20 transgenic mice after a total of three months of meningeal lymphatic ablation (Extended Data Fig. 9f); amyloid-β aggregates had formed in the meninges (Extended Data Fig. 9g) and the amyloid-β plaque load in the hippocampi of these mice was significantly increased (Extended Data Fig. 9h–k)

Evidence bc0dddfa00

Enrichment analysis revealed, however, changes in gene sets involved in immune and inflammatory responses, phospholipid metabolism, extracellular matrix organization, cellular adhesion and endothelial tube morphogenesis, all of which suggest that there are functional alterations in meningeal LECs with age (Fig. 2c).

Evidence 5098ac9a9a

Ageing also leads to progressive lymphatic vessel dysfunction in peripheral tissues

Evidence 406d64928e

Indeed, and in agreement with a previous study, old mice demonstrate reduced brain perfusion by CSF macromolecules compared to young counterparts (Extended Data Fig. 6a, b)

Evidence c64b793bc1

The altered expression of genes involved in the transmembrane receptor protein tyrosine kinase signalling pathway in old mice, namely the downregulation of Cdk5r1, Adamts3 and Fgfr3, indicated possible changes in signalling by lymphangiogenic growth factors in old meningeal LECs (Fig. 2d)

Evidence f5c8896d1d

Impaired brain perfusion by CSF in old mice was accompanied by a decrease in meningeal lymphatic vessel diameter and coverage, as well as decreased drainage of CSF macromolecules into dCLNs in both females and males (Extended Data Fig. 6c–f)

Evidence eaeee45204

Transcranial delivery (through a thinned skull surface) of hydrogel-encapsulated VEGF-C peptide also resulted in increased diameter of meningeal lymphatics in young and old mice (Extended Data Fig. 7a–c)

Evidence 04c47ae8cb

We have previously shown that treatment with recombinant VEGF-C increases the diameter of meningeal lymphatic vessels

Evidence 9cf3ea6f0f

Furthermore, delivery of VEGF-C by adenoviral gene therapy was previously found to efficiently boost peripheral lymphatic sprouting and function

Evidence 66b0aa7417

Treatment of young mice with AAV1-CM-mVEGF-C resulted in a significant increase in meningeal lymphatic vessel diameter, without affecting blood vessel coverage (Extended Data Fig. 6k–m)

Evidence c133280018

Treatment of old mice (at 20–24 months) with AAV1-CMV-mVEGF-C also resulted in increased lymphatic vessel diameter (compared to AAV1-CMV-eGFP) without detectable off-target effects on the meningeal blood vasculature coverage and on meningeal and/or brain vascular haemodynamics (Fig. 2e–h and Extended Data Fig. 6n–p)

Evidence eaadf93626

This VEGF-C treatment led to a significant increase in the function of meningeal lymphatic vessels in old mice, whereas young–adult mice did not respond to the treatment (Extended Data Fig. 7d, e), probably due to the ceiling effect of their existing capacity to drain OVA-A647

Evidence 71e07104df

Moreover, viral expression of mVEGF-C did not significantly affect the diameter of meningeal lymphatic vessels, the level of amyloid-β in the CSF, or amyloid-β deposition in the hippocampus (Extended Data Fig. 8g–n)

Evidence 9440436922

However, AAV1-CMV-mVEGF-C treatment resulted in significant improvement in the latency to platform and in the percentage of allocentric navigation strategies, in the MWM reversal at 12–14 months (Extended Data Fig. 7q, t) and in the MWM acquisition and reversal at 20–22 months (Extended Data Fig. 7r, u), compared to AAV1-CMV-eGFP-treated age-matched mice

Evidence d2d2b9976c

Increased expression of VEGF-C in the adult brain has previously been shown to boost proliferation of neural stem cells in the hippocampus

Evidence 4f1e00a266

The beneficial effect of mVEGF-C treatment in mice from the sham group, which performed significantly better in the NLR (Fig. 2n, o) and MWM (Fig. 2p–r) tests, was abrogated in mice in which the CSF-draining lymphatic vessels had been ligated

Evidence f5cb9e7825

Accordingly, the drainage of CSF macromolecules into dCLNs was significantly higher in sham-operated mice treated with mVEGF-C compared to all other groups (Fig. 2s, t)

Evidence 31e1df92b6

Independently of the model, the level of CSF tracer drained into the dCLNs was comparable between transgenic mice with Alzheimer’s disease and age-matched wild-type littermates (Extended Data Fig. 8p–s)

Evidence eb69ae0396

Similarly, the morphology and coverage of meningeal lymphatic vessels did not differ between wild-type and 5xFAD mice at 3–4 months of age (Extended Data Fig. 8t, u)

Evidence 80ec31b82c

Within the brain parenchyma, it was shown that aquaporin 4 (AQP4) expression by astrocytes plays an important role in the modulation of paravascular CSF macromolecule influx and efflux (through the glymphatic route)

Evidence be933189ef

Deletion of Aqp4 in transgenic mice with Alzheimer’s disease also resulted in increased amyloid-β plaque burden and exacerbated cognitive impairment

Evidence 3b0306ee7c

Treatment with VEGF-C156S resulted in a significant increase in meningeal lymphatic diameter (Extended Data Fig. 7i, j), drainage of tracer from the CSF (Extended Data Fig. 7k, l), and paravascular influx of tracer into the brains of old mice (Extended Data Fig. 7m, n)


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