Bogdan O. Popescu

Blood-brain barrier alterations in ageing and dementia

The current pathogenic scenarios of different types of dementia are based on a number of common mechanisms of neurodegeneration, such as accumulation of abnormal proteins (within or outside cells), mitochondrial dysfunction and oxidative stress, calcium homeostasis dysregulation, early synaptic disconnection and late apoptotic cell death. Ageing itself is associated with mild cognitive deterioration, probably due to subtle multifactorial changes resulting in a global decrease of a functional brain reserve. Increased age is a risk factor for neurodegeneration and key pathological features of dementia can also be found in aged brains. One of the underexplored brain structures in ageing and dementia is the blood-brain barrier (BBB), a complex cellular gate which regulates tightly the transport of molecules into and from the central nervous system. Disruption of this barrier is now increasingly documented not only in brain vascular disease but also in ageing and neurodegenerative disorders. To date, such evidence points mainly at an association between various dementia forms and disruption of the BBB. But, in reviewing such results, and taking into account the exquisite sensitivity of neuronal function to the composition of the interstitial brain fluid (IBF), which is regulated by the BBB, we would like to propose the existence of a possible causal link between alterations of BBB and conditions associated with cognitive decline.

Identification of telocytes in skeletal muscle interstitium: implication for muscle regeneration

Skeletal muscle interstitium is crucial for regulation of blood flow, passage of substances from capillaries to myocytes and muscle regeneration. We show here, probably, for the first time, the presence of telocytes (TCs), a peculiar type of interstitial (stromal) cells, in rat, mouse and human skeletal muscle. TC features include (as already described in other tissues) a small cell body and very long and thin cell prolongations—telopodes (Tps) with moniliform appearance, dichotomous branching and 3D‐network distribution. Transmission electron microscopy (TEM) revealed close vicinity of Tps with nerve endings, capillaries, satellite cells and myocytes, suggesting a TC role in intercellular signalling (via shed vesicles or exosomes). In situ immunolabelling showed that skeletal muscle TCs express c‐kit, caveolin‐1 and secrete VEGF. The same phenotypic profile was demonstrated in cell cultures. These markers and TEM data differentiate TCs from both satellite cells (e.g. TCs are Pax7 negative) and fibroblasts (which are c‐kit negative). We also described non‐satellite (resident) progenitor cell niche. In culture, TCs (but not satellite cells) emerge from muscle explants and form networks suggesting a key role in muscle regeneration and repair, at least after trauma.

Telocytes in meninges and choroid plexus.

Telocytes (TCs) are a recently identified type of interstitial cells present in a wide variety of organs in humans and mammals (www.telocytes.com). They are characterized by a small cell body, but extremely long cell processes - telopodes (Tp), and a specific phenotype. TCs establish close contacts with blood capillaries, nerve fibers and stem cells. We report here identification of TCs by electron microscopy and immunofluorescence in rat meninges and choroid plexus/subventricular zone, in the vicinity of putative stem cells. The presence of TCs in brain areas involved in adult neurogenesis might indicate that they have a role in modulation of neural stem cell fate.

Cellular Players in Skeletal Muscle Regeneration

Skeletal muscle, a tissue endowed with remarkable endogenous regeneration potential, is still under focused experimental investigation mainly due to treatment potential for muscle trauma and muscular dystrophies. Resident satellite cells with stem cell features were enthusiastically described quite a long time ago, but activation of these cells is not yet controlled by any medical interventions. However, after thorough reports of their existence, survival, activation, and differentiation there are still many questions to be answered regarding the intimate mechanism of tissue regeneration. This review delivers an up-to-date inventory of the main known key players in skeletal muscle repair, revealed by various models of tissue injuries in mechanical trauma, toxic lesions, and muscular dystrophy. A better understanding of the spatial and temporal relationships between various cell populations, with different physical or paracrine interactions and phenotype changes induced by local or systemic signalling, might lead to a more efficient approach for future therapies.

Triggers and Effectors of Oxidative Stress at Blood-Brain Barrier Level: Relevance for Brain Ageing and Neurodegeneration

As fundamental research advances, it is becoming increasingly clear that a clinically expressed disease implies a mixture of intertwining molecular disturbances. Oxidative stress is one of such pathogenic pathways involved in virtually all central nervous system pathologies, infectious, inflammatory, or degenerative in nature. Since brain homeostasis largely depends on integrity of blood-brain barrier (BBB), many studies focused lately on BBB alteration in a wide spectrum of brain diseases. The proper two-way molecular transfer through BBB depends on several factors, including the functional status of its tight junction (TJ) complexes of proteins sealing neighbour endothelial cells. Although there is abundant experimental work showing that oxidative stress associates BBB permeability alteration, less is known about its implications, at molecular level, in TJ protein expression or TJ-related cell signalling. In this paper, oxidative stress is presented as a common pathway for different brain pathogenic mechanisms which lead to BBB dysregulation. We revise here oxidative-induced molecular mechanisms of BBB disruption and TJ protein expression alteration, in relation to ageing and neurodegeneration.

Platelet‐derived growth factor receptor‐β‐positive telocytes in skeletal muscle interstitium

Telocytes (TCs) represent a new cell type recently described in mammalian skeletal muscle interstitium as well as in other organs. These have a specific morphology and phenotype, both in situ and in vitro. Telocytes are cells with long and slender cell prolongations, in contact with other interstitial cells, nerve fibres, blood capillaries and resident stem cells in niches. Our aim was to investigate the potential contribution of TCs to micro‐vascular networks by immunofluorescent labelling of specific angiogenic growth factors and receptors. We found that in human skeletal muscle TCs were constantly located around intermediate and small blood vessels and endomysial capillaries. Epi‐fluorescence and laser confocal microscopy showed that TCs express c‐kit, platelet‐derived growth factor receptor (PDGFR)‐β and VEGF, both in situ and in vitro. Telocytes were constantly located in the perivascular or pericapillary space, as confirmed by double staining of c‐kit/CD31, PDGFR‐β/CD31 and PDGFR‐β/α‐smooth muscle actin, respectively. Electron microscopy (EM) differentiated between pericytes and other cell types. Laminin labelling showed that TCs are not enclosed or surrounded by a basal lamina in contrast to mural cells. In conclusion, a) PDGFR‐β could be used as a marker for TCs and b) TCs are presumably a transitional population in the complex process of mural cell recruitment during angiogenesis and vascular remodelling.

Vascular cognitive impairment, dementia, aging and energy demand. A vicious cycle

To a great extent, cognitive health depends on cerebrovascular health and a deeper understanding of the subtle interactions between cerebrovascular function and cognition is needed to protect humans from one of the most devastating affliction, dementia. However, the underlying biological mechanisms are still not completely clear. Many studies demonstrated that the neurovascular unit is compromised in cerebrovascular diseases and also in other types of dementia. The hemodynamic neurovascular coupling ensures a strong increase of the cerebral blood flow (CBF) and an acute increase in neuronal glucose uptake upon increased neural activity. Dysfunction of cerebral autoregulation with increasing age along with age-related structural and functional alterations in cerebral blood vessels including accumulation of amyloid-beta (Aβ) in the media of cortical arterioles, neurovascular uncoupling due to astrocyte endfeet retraction, impairs the CBF and increases the neuronal degeneration and susceptibility to hypoxia and ischemia. A decreased cerebral glucose metabolism is an early event in Alzheimer’s disease (AD) pathology and may precede the neuropathological Aβ deposition associated with AD. Aβ accumulation in turn leads to further decreases in the CBF closing the vicious cycle. Alzheimer, aging and diabetes are also influenced by insulin/insulin-like growth factor-1 signaling, and accumulated evidence indicates sporadic AD is associated with disturbed brain insulin metabolism. Understanding how vascular and metabolic factors interfere with progressive loss of functional neuronal networks becomes essential to develop efficient drugs to prevent cognitive decline in elderly.

Dantrolene protects neurons against kainic acid induced apoptosis in vitro and in vivo

Apoptotic cell death induced by kainic acid (KA) in cultures of rat cerebellar granule cells (CGC) and in different brain regions of Wistar rat pups on postnatal day 21 (P21) was studied. In vitro, KA (100–500 μM) induced a concentration‐dependent loss of cell viability in MTT assay and cell death had apoptotic morphology as studied by chromatin staining with propidium iodide (PI). In vivo, twenty‐four hours after induction of status epilepticus (SE) by an intraperitoneal KA injection (5 mg/kg) we quantified apoptotic cells in hippocampus (CA1 and CA3), parietal cortex and cerebellum using PI staining and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL) technique. We report that dantrolene, a specific ryanodine receptor antagonist, was able to significantly reduce the apoptotic cell death in CGC cultures and in hyppocampal CA1 and parietal cortex regions. Our finding can be valuable for neuroprotective therapy strategies in patients with repeated generalized seizures or status epilepticus.

Altered expression of claudin family proteins in Alzheimer’s disease and vascular dementia brains

Claudins (Cls) are a multigene family of transmembrane proteins with different tissue distribution, which have an essential role in the formation and sealing capacity of tight junctions (TJs). At the level of the blood–brain barrier (BBB), TJs are the main molecular structures which separate the neuronal milieu from the circulatory space, by a restriction of the paracellular flow of water, ions and larger molecules into the brain. Different studies suggested recently significant BBB alterations in both vascular and degenerative dementia types. In a previous study we found in Alzheimer’s disease (AD) and vascular dementia (VaD) brains an altered expression of occludin, a molecular partner of Cls in the TJs structure. Therefore in this study, using an immunohistochemical approach, we investigated the expression of Cl family proteins (Cl‐2, Cl‐5 and Cl‐11) in frontal cortex of aged control, AD and VaD brains. To estimate the number of Cl‐expressing cells, we applied a random systematic sampling and the unbiased optical fractionator method. We found selected neurons, astrocytes, oligodendrocytes and endothelial cells expressing Cl‐2, Cl‐5 and Cl‐11 at detectable levels in all cases studied. We report a significant increase in ratio of neurons expressing Cl‐2, Cl‐5 and Cl‐11 in both AD and VaD as compared to aged controls. The ratio of astrocytes expressing Cl‐2 and Cl‐11 was significantly higher in AD and VaD as compared to aged controls. The ratio of oligodendrocytes expressing Cl‐11 was significantly higher in AD and the ratio of oligodendrocytes expressing Cl‐2 was significantly higher in VaD as compared to aged controls. Within the cerebral cortex, Cls were selectively expressed by pyramidal neurons, which are the ones responsible for cognitive processes and affected by AD pathology. Our findings suggest a new function of Cl family proteins which might be linked to response to cellular stress.

Occludin is overexpressed in Alzheimer's disease and vascular dementia

The tight junctions (TJs) are key players in the control of blood‐brain barrier (BBB) properties, the most complex TJs in the vascular system being found in the endothelial cells of brain capillaries. One of the main TJs proteins is occludin, which anchors plasma membranes of neighbour cells and is present in large amounts in the brain endothelia. Previous studies demonstrated that disruption of BBB in various pathological situations associates with changes in occludin expression, and this change could be responsible for malfunction of BBB. Therefore in this study, applying an immunohistochemical approach, we decided to explore the occludin expression in frontal cortex (FC) and basal ganglia in ageing control, Alzheimers disease (AD), and vascular dementia (VD) brains, as far as all these pathologies associate microangiopathy and disruption of BBB. Strikingly, we found selected neurons, astrocytes and oligodendrocytes expressing occludin, in all cases studied. To estimate the number of occludin‐expressing neurons, we applied a stereological approach with random systematic sampling and the unbiased optical fractionator method. We report here a significant increase in ratio of occludin‐expressing neurons in FC and basal ganglia regions in both AD and VD as compared to ageing controls. Within the cerebral cortex, occludin was selectively expressed by pyramidal neurons, which are the ones responsible for cognitive processes and affected by AD pathology. Our findings could be important in unravelling new pathogenic pathways in dementia disorders and new functions of occludin and TJs.