Posts Tagged ‘β2-microglobulin’

impairment of cognitive function and neurogenesis

Larry H. Bernstein, MD, FCAP, Curator



β2-microglobulin is a systemic pro-aging factor that impairs cognitive function and neurogenesis

Lucas K SmithYingbo HeJeong-Soo ParkGregor BieriCedric E SnethlageKarin LinGeraldine GontierRafael Wabl, et al.
Nature Medicine 21,932–937(2015)

Aging drives cognitive and regenerative impairments in the adult brain, increasing susceptibility to neurodegenerative disorders in healthy individuals1, 2, 3, 4. Experiments using heterochronic parabiosis, in which the circulatory systems of young and old animals are joined, indicate that circulating pro-aging factors in old blood drive aging phenotypes in the brain5, 6. Here we identify β2-microglobulin (B2M), a component of major histocompatibility complex class 1 (MHC I) molecules, as a circulating factor that negatively regulates cognitive and regenerative function in the adult hippocampus in an age-dependent manner. B2M is elevated in the blood of aging humans and mice, and it is increased within the hippocampus of aged mice and young heterochronic parabionts. Exogenous B2M injected systemically, or locally in the hippocampus, impairs hippocampal-dependent cognitive function and neurogenesis in young mice. The negative effects of B2M and heterochronic parabiosis are, in part, mitigated in the hippocampus of young transporter associated with antigen processing 1 (Tap1)-deficient mice with reduced cell surface expression of MHC I. The absence of endogenous B2M expression abrogates age-related cognitive decline and enhances neurogenesis in aged mice. Our data indicate that systemic B2M accumulation in aging blood promotes age-related cognitive dysfunction and impairs neurogenesis, in part via MHC I, suggesting that B2M may be targeted therapeutically in old age.

Figure 1: Systemic B2M increases with age and impairs hippocampal-dependent cognitive function and neurogenesis

Systemic B2M increases with age and impairs hippocampal-dependent cognitive function and neurogenesis.

(a,b) Schematics of unpaired young versus aged mice (a), and young isochronic versus heterochronic parabionts (b). (a,b) Changes in plasma concentration of B2M with age at 3, 6, 12, 18 and 24 months (a) and between young isochronic and…


Figure 2: B2M expression increases in the aging hippocampus and impairs hippocampal-dependent cognitive function and neurogenesis.close

B2M expression increases in the aging hippocampus and impairs hippocampal-dependent cognitive function and neurogenesis.

(a,b) Western blot and quantification of hippocampal lysates probed with B2M- and actin-specific antibodies from young (3 months) and aged (18 months) unpaired animals (a), or young isochronic and young heterochronic parabionts five wee…

Figure 3: Reducing MHC I surface expression mitigates the negative effects of heterochronic parabiosis on neurogenesis.close

Reducing MHC I surface expression mitigates the negative effects of heterochronic parabiosis on neurogenesis.

(a) Schematic of young (3 months) WT and Tap1−/− isochronic parabionts and young WT and Tap1−/− heterochronic parabionts. (b,c) Representative (of six sections per mouse) images of the DG (b) and quantification of DCX immunostaining (c)…


Figure 4: Absence of B2M enhances hippocampal-dependent cognitive function and neurogenesis in aged animals.

Absence of B2M enhances hippocampal-dependent cognitive function and neurogenesis in aged animals.

(ad) Learning and memory in young (3 months) and aged (17 months) WT and B2m-knockout (B2m−/−) mice by RAWM (a,c) and contextual fear conditioning (b,d). Data are from 10 young WT, 10 young B2m−/−, 8 aged WT, and 12 aged B2m−/− mice. (…


Neuroscience. 2015 Nov 12;308:75-94. doi: 10.1016/j.neuroscience.2015.09.012. Epub 2015 Sep 10.
Synergistic neuroprotection by epicatechin and quercetin: Activation of convergent mitochondrial signaling pathways.
In view of evidence that increased consumption of epicatechin (E) and quercetin (Q) may reduce the risk of stroke, we have measured the effects of combining E and Q on mitochondrial function and neuronal survival following oxygen-glucose deprivation (OGD). Relative to mouse cortical neuron cultures pretreated (24h) with either E or Q (0.1-10μM), E+Q synergistically attenuated OGD-induced neuronal cell death. E, Q and E+Q (0.3μM) increased spare respiratory capacity but only E+Q (0.3μM) preserved this crucial parameter of neuronal mitochondrial function after OGD. These improvements were accompanied by corresponding increases in cyclic AMP response element binding protein (CREB) phosphorylation and the expression of CREB-target genes that promote neuronal survival (Bcl-2) and mitochondrial biogenesis (PGC-1α). Consistent with these findings, E+Q (0.1 and 1.0μM) elevated mitochondrial gene expression (MT-ND2 and MT-ATP6) to a greater extent than E or Q after OGD. Q (0.3-3.0μM), but not E (3.0μM), elevated cytosolic calcium (Ca(2+)) spikes and the mitochondrial membrane potential. Conversely, E and E+Q (0.1 and 0.3μM), but not Q (0.1 and 0.3μM), activated protein kinase B (Akt). Nitric oxide synthase (NOS) inhibition with L-N(G)-nitroarginine methyl ester (1.0μM) blocked neuroprotection by E (0.3μM) or Q (1.0μM). Oral administration of E+Q (75mg/kg; once daily for 5days) reduced hypoxic-ischemic brain injury. These findings suggest E and Q activate Akt- and Ca(2+)-mediated signaling pathways that converge on NOS and CREB resulting in synergistic improvements in neuronal mitochondrial performance which confer profound protection against ischemic injury.
MiR-34a regulates blood–brain barrier permeability and mitochondrial function by targeting cytochrome c



The blood–brain barrier is composed of cerebrovascular endothelial cells and tight junctions, and maintaining its integrity is crucial for the homeostasis of the neuronal environment. Recently, we discovered that mitochondria play a critical role in maintaining blood–brain barrier integrity. We report for the first time a novel mechanism underlying blood–brain barrier integrity: miR-34a mediated regulation of blood–brain barrier through a mitochondrial mechanism. Bioinformatics analysis suggests miR-34a targets several mitochondria-associated gene candidates. We demonstrated that miR-34a triggers the breakdown of blood–brain barrier in cerebrovascular endothelial cell monolayer in vitro, paralleled by reduction of mitochondrial oxidative phosphorylation and adenosine triphosphate production, and decreased cytochrome c levels.


The blood–brain barrier (BBB) is composed of highly specialized cerebrovascular endothelial cells (CECs), separates brain tissue from the circulating blood, and maintains homeostasis of the neuronal environment.1 The CECs are interconnected by tight junctions including cytoplasmic zonula occludens (ZO) proteins, and various transmembrane proteins such as occludin and claudins.2 Disruption of BBB tight junctions has been well documented in cerebrovascular diseases and neurodegenerative disorders and is considered to be a pathological condition of the diseases and plays a key role in disease progression as well.2

A recent study demonstrates that the mitochondrial mechanisms regulate BBB integrity and permeability using oxygen–glucose deprivation and reoxygenation (OGD-R), anin vitro model of ischemic reperfusion injury.3 Our work demonstrates that compromised mitochondria lead to the disruption of tight junctions, opening of the BBB, and exacerbation of stroke outcomes.4 As such, regulation of mitochondrial function may affect BBB openings and could be critical in limiting the pathological progression of cerebrovascular diseases and neurodegenerative disorders.

MicroRNAs (miRNAs) are short non-coding functional RNAs that target certain messenger RNAs (mRNAs) through complementary base-pairing between the miRNAs and its mRNA targets, resulting in the inhibition of mRNA translation or degradation of mRNA.5 It has been documented that miRNAs are involved in mitochondrial structure and function, such as miR-181c which regulates mitochondrial morphology,6 miR-1 which affects mitochondrial mRNA translation,7 and miR-378 which targets mitochondrial enzymes involved in oxidative energy metabolism.8 Additionally, several miRNAs have recently been found to regulate BBB permeability. MiR-155, miR-181c, and miR-29c negatively affect BBB function by targeting tight junction protein genes directly or affecting related signal pathways.911 The miR-34 family members were discovered computationally and later verified experimentally as a part of the p53 tumor suppressor network. Recent work demonstrates that miR-34a modulates the expression of synaptic targets and neuronal morphology and function.12 However, little is known regarding the role of miR-34a in mitochondrial function and BBB permeability.

In the present study, we report that the overexpression of miR-34a breaks down the BBB through inhibition of mitochondrial function. Furthermore, cytochrome c (CYC) is experimentally verified as a target of miR-34a in vitro.


Overexpression of miR-34a affects BBB permeability and disrupts tight junctions in CECs

To determine whether miR-34a functionally affected the BBB, we transfected CECs with miR34a plasmid versus vector control in 24-well plates, cultured the cells for 48 h, conducted a BBB permeability assay in a CEC monolayer transwell system in vitro with an additional culture of 48 h, and measured the fluorescent dye FD-4 permeability of each well (Figure 1(a)). As shown in Figure 1(a), FD-4 permeability was significantly increased in wells containing miR-34a overexpression CEC monolayer. Papp, the permeability coefficient, was also significantly higher in CECs overexpressed with miR-34a in comparison to vector controls (Figure 1(a)). Furthermore, immunohis-tochemistry staining of tight junction-related proteins revealed that ZO-1 was continuously distributed in the control, but a discontinuous distribution of ZO-1 was observed in miR-34a overexpressed CEC monolayer (Figure 1(b)). Disruption of tight junctions was not associated with cell viability in CECs transfected with plasmids for 48 h or 96 h (Supplementary Figure 2). Altogether, these data suggest that overexpression of miR-34a increases BBB permeability and compromises BBB tight junctions.

Figure 1.

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Figure 1.

Overexpression of miR-34a increases BBB permeability in vitro. (a) A schematic protocol using fluorescein isothiocyanate–dextran-4 (FD-4) to detect BBB permeability in vitro. FD-4 permeability in CECs that overexpressed miR-34a plasmid (0.017 ng) versus control was presented as real-time rate of FD-4 mean fluorescent intensity (2-way ANOVA followed by post hoc Dunnett’s test; n = 3; **, P < 0.01; ****, P < 0.0001). Calculated apparent permeability coefficient Papp(Student’s t-test; ****, P < 0.0001) is expressed as mean ± SD. (b) Confocal fluorescence images of CECs confluent monolayers confirmed microscopically after transfection with miR-34a plasmid versus control. Fluorescent staining: tight junctions ZO-1 (red), cell nuclei (DAPI, blue). Overexpression of miR-34a apparently disrupted tight junctions and resulted in gaps between cells (white arrows). Results are representative of three independent experiments.

MiR-34a affects mitochondrial function by targeting CYC in CECs

Our recent work demonstrated that mitochondria play a pivotal role in the maintenance of BBB integrity. BBB tight junctions are rapidly disrupted if oxidative phosphorylation is reduced by mitochondrial inhibitors.4 To investigate whether the miR-34a regulates BBB openings via affecting mitochondrial function in CECs, we examined cellular energetic OCRs in CECs transfected with miR-34a plasmid versus vector control. Interestingly, overexpression of miR-34a significantly impaired mitochondrial function in CECs (Figure 2(a) and Supplementary Figure 3). Basal respiration, ATP production, maximal respiration, and spare capacity were all significantly reduced in CECs overexpressing miR-34a for 48 and 72 h (Figure 2(a)). ATP level was also substantially reduced in CECs following overexpression of miR-34a in a dose dependent manner at 72 h (Figure 2(b)).

Figure 2.

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Figure 2.

Overexpression of mir-34a reduces mitochondrial function and decreases CYC level in cerebrovascular endothelial cells. (a) Basal respiration, ATP production, maximal respiration, and spare capacity were calculated from the bioenergetics functional assay at post-transfection 48 and 72 h (raw data in Supplementary Figure 3). Data are expressed as mean ± SD (n = 5). 1-way ANOVA followed by post hoc Tukey’s test. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (b) ATP level was measured at 72 h post-transfection. Data are expressed as mean ± SD (n = 5). 1-way ANOVA followed by post hoc Tukey’s test. (****, P < 0.0001). (c) Bioinfomatic analysis of miR-34a-targeting candidates related to mitochondria. (d) Flow cytometry analysis of mitochondrial specific proteins for complex I proteins (NDUFAF1, NDUFC2 and NDUFS2), complex II protein (SDHC), complex III protein (CYB), complex IV protein (CYC oxidase, Cox IV), cytochrome c (CYCS), pyruvate dehydrogenase kinase (PDK), and voltage-dependent anion channel protein (VDAC) at 72 h post-transfection. CYC level was significantly lower in the cells that were transfected with the miR-34a plasmid. Data are presented as mean ± SD (n = 3) and analyzed by Student’s t-test, *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Results are representative of three independent experiments.

To further determine miR-34a targets and uncover the mechanism that is used to affect mitochondria, we performed a bioinformatics analysis of the miR-34a database (miRbase and TargetScan). MiR-34a potentially targets several mitochondria-associated gene candidates including succinate dehydrogenase subunit c (SDHC), cytochrome B reductase 1 (CYBRD1), cytochrome B5 reductase 3 (CYBRD5), cytochrome c (CYCS), pyruvate dehydrogenase kinase isozyme 1 and 2 (PDK1 and PDK2) (Figure 2(c). However, CECs transfected with the miR-34a plasmid had robustly decreased CYCS levels measured by flow cytometry, suggesting that CYCS is one of the miR-34a targets among the potential candidates (Figure 2(d)). Moreover, overexpression of miR-34a slightly increased potential target SDHC but did not change the protein level of CYB and PKD (Figure 2(d)). Off-target genes, NDUFAF1, and VDAC showed no significant change in protein level, but NDUFC2, NDUFS2, and Cox IV were all increased in parallel with overexpression of miR-34a (Figure 2(d)). Taken together, these results experimentally verified CYCS as a miR-34a target, which is associated with the reduction of mitochondrial oxidative phosphorylation in CECs.


In the present study, we demonstrated that the overexpression of miR-34a results in an increased BBB permeability and the disruption of tight junctions ZO-1 in CECs. Consistently, overexpression of miR-34a impaired mitochondrial oxidative phosphorylation and reduced ATP production in CECs. Bioinformatics analysis revealed series of potential miR-34a-targeting candidates related to mitochondrial function. We elucidated that CYCS is a miR-34a target, and the overexpression of miR-34a inhibited the CYCS expression and increased with the expression of other mitochondria-associated genes.

The overexpression of miR-34a disrupted tight junction protein ZO-1 (Figure 1). However, bioinformatics analysis indicated that miR-34a did not target the ZO-1 gene or other tight junction related genes, which suggests that the increased BBB permeability is not directly caused by the targeting of tight junction protein genes. The compromised mitochondrial function by overexpression of miR-34a may influence cellular metabolism in a way that is critical to maintain BBB tight junctions. Among several potential mitochondria-associated gene targets (Figure 2(c)), miR-34a initiated the reduction of CYCS level. Interestingly, potential target SDHC and other off-target gene proteins (NDUFC2, NDUFS2, and Cox IV) were concurrently upregulated (Figure 2(d)), which might be due to the compensation for the reduced target gene protein CYCS, or the disturbance of the coordinated gene translation in mitochondria. We therefore concluded that CYCS is a miR-34a target and is responsible for the miR-34a-induced reduction of mitochondrial oxidative phosphorylation.

Protein kinase C (PKC) signaling has also been shown to affect BBB or other endothelial barriers in vitro and in vivo. A recent study reported that miR-34a regulated blood–tumor barrier by targeting PKCɛ using glioma endothelial cells.13 In this study, we did not assess the PKC pathways that could contain additional targets of miR-34a. However, our data do support that miR-34a affects BBB via a mitochondrial mechanism, which is novel and may lead a new direction for designing BBB-related therapeutics.

We have noted several limitations in our study. First, we did not examine the effects of knockdown or knockout miR-34a on BBB function, which might fully establish the role of miR-34a in the BBB and mitochondria. Second, this work was conducted in cell culture models, which adequately address the mechanism of effect that miR-34a exerts on the BBB and mitochondria but do not provide evidence of its involvement in cerebrovascular or neurodegenerative conditions. Further studies in relevant experimental models are warranted.

Mitochondria play a pivotal role in cellular bioenergetics and cell survival, participating in a variety of cellular processes, including the generation of ATP, and the regulation of apoptotic signaling and other signaling pathways.14 MiR-34a targets and represses multiple genes involved in cell proliferation, apoptosis, cell cycle, migration, etc.,15 but it is not known if these effects are modulated by the observed mitochondrial effects as well. The present study provides the first description of miR-34a affecting mitochondrial activity, which could lead to a revision of current miR-34a targets and may lead to discovery of new mechanisms. The elucidation of the miR-34a’s role in mitochondrial oxidative phosphorylation and the BBB integrity offers a novel therapeutic strategy for targeting miR-34a to treat cerebrovascular and neurodegenerative diseases such as stroke and Alzheimer’s disease. These neuropathological diseases are known to involve a host of conditions that lead to mitochondrial impairment and BBB disruption. Finally, transient opening of the BBB could prove to be useful for CNS drug delivery.


Long-term aerobic exercise prevents age-related brain deterioration

October 30, 2015

A study of the brains of mice shows that structural deterioration associated with old age can be prevented by long-term aerobic exercise starting in mid-life, according to the authors of an open-access paper in the journal PLOS Biologyyesterday (October 29).

Old age is the major risk factor for Alzheimer’s disease, like many other diseases, as the authors at The Jackson Laboratory in Bar Harbor, Maine, note. Age-related cognitive deficits are due partly to changes in neuronal function, but also correlate with deficiencies in the blood supply to the brain and with low-level inflammation.

“Collectively, our data suggests that normal aging causes significant dysfunction to the cortical neurovascular unit, including basement membrane reduction and pericyte (cells that wrap around blood capillaries) loss. These changes correlate strongly with an increase in microglia/monocytes in the aged cortex,” said Ileana Soto, lead author on the study.*

Benefits of aerobic exercise

However, the researchers found that if they let the mice run freely, the structural changes that make the blood-brain barrier leaky and result in inflammation of brain tissues in old mice can be mitigated. That suggests that there are also beneficial effects of exercise on dementia in humans.**

Further work will be required to establish the mechanism(s): what is the role of the complement-producing microglia/macrophages, how does Apoe decline contribute to age-related neurovascular decline, does the leaky blood-brain barrier allow the passage of damaging factors from the circulation into the brain?

This work was funded in part by The Jackson Laboratory Nathan Shock Center, the Fraternal Order of the Eagle, the Jane B Cook Foundation and NIH.

* The authors investigated the changes in the brains of normal young and aged laboratory mice by comparing by their gene expression profiles using a technique called RNA sequencing, and by comparing their structures at high-resolution by using fluorescence microscopy and electron microscopy. The gene expression analysis indicated age-related changes in the expression of genes relevant to vascular function (including focal adhesion, vascular smooth muscle and ECM-receptor interactions), and inflammation (especially related to the complement system, which clears foreign particles) in the brain cortex.

These changes were accompanied by a decline in the function of astrocytes (key support cells in the brain) and loss of pericytes (the contractile cells that surround small capillaries and venules and maintain the blood-brain barrier). There were also effects on the basement membrane, which forms an integral part of the blood-brain barrier, as well as an increase in the density and functional activation of the immune cells known as microglia/monocytes, which scavenge the brain for infectious agents and damaged cells.

** To investigate the impact of long-term physical exercise on the brain changes seen in the aging mice, the researchers provided the animals with a running wheel from 12 months old (equivalent to middle aged in humans) and assessed their brains at 18 months (equivalent to ~60yrs old in humans, when the risk of Alzheimer’s disease is greatly increased). Young and old mice alike ran about two miles per night, and this physical activity improved the ability and motivation of the old mice to engage in the typical spontaneous behaviors that seem to be affected by aging.

This exercise significantly reduced age-related pericyte loss in the brain cortex and improved other indicators of dysfunction of the vascular system and blood-brain barrier. Exercise also decreased the numbers of microglia/monocytes expressing a crucial initiating component of the complement pathway that others have shown previously to play are role in age-related cognitive decline. Interestingly, these beneficial effects of exercise were not seen in mice deficient in a gene called Apoe, variants of which are a major genetic risk factor for Alzheimer’s disease. The authors also report that Apoe expression in the brain cortex declines in aged mice and this decline can also be prevented by exercise.

Abstract of APOE Stabilization by Exercise Prevents Aging Neurovascular Dysfunction and Complement Induction

Aging is the major risk factor for neurodegenerative diseases such as Alzheimer’s disease, but little is known about the processes that lead to age-related decline of brain structures and function. Here we use RNA-seq in combination with high resolution histological analyses to show that aging leads to a significant deterioration of neurovascular structures including basement membrane reduction, pericyte loss, and astrocyte dysfunction. Neurovascular decline was sufficient to cause vascular leakage and correlated strongly with an increase in neuroinflammation including up-regulation of complement component C1QA in microglia/monocytes. Importantly, long-term aerobic exercise from midlife to old age prevented this age-related neurovascular decline, reduced C1QA+ microglia/monocytes, and increased synaptic plasticity and overall behavioral capabilities of aged mice. Concomitant with age-related neurovascular decline and complement activation, astrocytic Apoe dramatically decreased in aged mice, a decrease that was prevented by exercise. Given the role of APOE in maintaining the neurovascular unit and as an anti-inflammatory molecule, this suggests a possible link between astrocytic Apoe, age-related neurovascular dysfunction and microglia/monocyte activation. To test this, Apoe-deficient mice were exercised from midlife to old age and in contrast to wild-type (Apoe-sufficient) mice, exercise had little to no effect on age-related neurovascular decline or microglia/monocyte activation in the absence of APOE. Collectively, our data shows that neurovascular structures decline with age, a process that we propose to be intimately linked to complement activation in microglia/monocytes. Exercise prevents these changes, but not in the absence of APOE, opening up new avenues for understanding the complex interactions between neurovascular and neuroinflammatory responses in aging and neurodegenerative diseases such as Alzheimer’s disease.


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