Chronic Hippocampal Expression of Notch Intracellular Domain Induces Vascular Thickening, Reduces Glucose Availability, and Exacerbates Spatial Memory Deficits in a Rat Model of Early Alzheimer.

The specific roles of Notch in progressive adulthood neurodegenerative disorders have begun to be unraveled in recent years. A number of independent studies have shown significant increases of Notch expression in brains from patients at later stages of sporadic Alzheimer's disease (AD). However, the impact of Notch canonical signaling activation in the pathophysiology of AD is still elusive. To further investigate this issue, 2-month-old wild-type (WT) and hemizygous McGill-R-Thy1-APP rats (Tg(+/-)) were injected in CA1 with lentiviral particles (LVP) expressing the transcriptionally active fragment of Notch, known as Notch Intracellular Domain (NICD), (LVP-NICD), or control lentivirus particles (LVP-C). The Tg(+/-) rat model captures presymptomatic aspects of the AD pathology, including intraneuronal amyloid beta (Aß) accumulation and early cognitive deficits. Seven months after LVP administration, Morris water maze test was performed, and brains isolated for biochemical and histological analysis. Our results showed a learning impairment and a worsening of spatial memory in LVP-NICD- as compared to LVP-C-injected Tg(+/-) rats. In addition, immuno histochemistry, ELISA multiplex, Western blot, RT-qPCR, and 1H-NMR spectrometry of cerebrospinal fluid (CSF) indicated that chronic expression of NICD promoted hippocampal vessel thickening with accumulation of Aß in brain microvasculature, alteration of blood-brain barrier (BBB) permeability, and a decrease of CSF glucose levels. These findings suggest that, in the presence of early Aß pathology, expression of NICD may contribute to the development of microvascular abnormalities, altering glucose transport at the BBB with impact on early decline of spatial learning and memory.

Deciphering variability in the role of interleukin-1ß in Parkinson's disease.

Although the role of inflammation in neurodegeneration has been well acknowledged, less is known on the issue of each cytokine in specific neurodegenerative diseases. In this review, we will present evidence elucidating that interleukin-1ß (IL-1ß) has a multi-faceted character in pathogenesis of Parkinson's disease, which is a progressive neurodegenerative disorder. Increased levels of IL-1ß were found in PD patients. Besides, PD symptoms were observed in IL-1ß wild-type, but not deficient, animals. These lines of evidence suggest that IL-1ß may contribute to the initiation or progression of PD. On the other hand, some studies reported decreased levels of IL-1ß in PD patients. Also, genetic studies provided evidence suggesting that IL-1ß may protect individuals against PD. Presumably, the broad range of IL-1ß role is due to its interaction with both upstream and downstream mediators. Differences in IL-1ß levels could be because of glia population (i.e. microglia and astrocytes), mitogen-activated protein kinase and nuclear factor κ light-chain-enhancer of activated B cells signaling pathways, and several mediators (including cyclooxygenase, neurotrophic factors, reactive oxygen species, caspases, heme oxygenase-1, and matrix metalloproteinases). Although far from practice at this point, unraveling theoretical therapeutic targets based on the up-down IL-1ß neuroweb could facilitate the development of strategies that are likely to be used for pharmaceutical designs of anti-neurodegenerative drugs of the future.

Glial Cell-Elicited Activation of Brain Microvasculature in Response to Brucella abortus Infection Requires ASC Inflammasome-Dependent IL-1ß Production.

Blood-brain barrier activation and/or dysfunction are a common feature of human neurobrucellosis, but the underlying pathogenic mechanisms are largely unknown. In this article, we describe an immune mechanism for inflammatory activation of human brain microvascular endothelial cells (HBMEC) in response to infection with Brucella abortus Infection of HBMEC with B. abortus induced the secretion of IL-6, IL-8, and MCP-1, and the upregulation of CD54 (ICAM-1), consistent with a state of activation. Culture supernatants (CS) from glial cells (astrocytes and microglia) infected with B. abortus also induced activation of HBMEC, but to a greater extent. Although B. abortus-infected glial cells secreted IL-1ß and TNF-α, activation of HBMEC was dependent on IL-1ß because CS from B. abortus-infected astrocytes and microglia deficient in caspase-1 and apoptosis-associated speck-like protein containing a CARD failed to induce HBMEC activation. Consistently, treatment of CS with neutralizing anti-IL-1ß inhibited HBMEC activation. Both absent in melanoma 2 and Nod-like receptor containing a pyrin domain 3 are partially required for caspase-1 activation and IL-1ß secretion, suggesting that multiple apoptosis-associated speck-like protein containing CARD-dependent inflammasomes contribute to IL-1ß-induced activation of the brain microvasculature. Inflammasome-mediated IL-1ß secretion in glial cells depends on TLR2 and MyD88 adapter-like/TIRAP. Finally, neutrophil and monocyte migration across HBMEC monolayers was increased by CS from Brucella-infected glial cells in an IL-1ß-dependent fashion, and the infiltration of neutrophils into the brain parenchyma upon intracranial injection of B. abortus was diminished in the absence of Nod-like receptor containing a pyrin domain 3 and absent in melanoma 2. Our results indicate that innate immunity of the CNS set in motion by B. abortus contributes to the activation of the blood-brain barrier in neurobrucellosis and IL-1ß mediates this phenomenon.

Influence of Peripheral inflammation on the progression of multiple sclerosis: evidence from the clinic and experimental animal models.

Multiple sclerosis (MS) is a chronic inflammatory disease characterized by demyelination, remyelination and loss of functions. Even though its etiology is unknown viral, genetic and environmental factors are considered triggers of the disease. MS shows a heterogeneous clinical course, but most patients exhibit exacerbations and remissions from the onset, eventually leading to secondary progressive multiple sclerosis. Systemic inflammatory events are known to signal into the central nervous system (CNS), and can induce a general response known as sickness behavior. Several research papers have demonstrated that a peripheral stimulus can induce the synthesis of cytokines in the brain. In different neurodegenerative diseases peripheral inflammation generates exacerbation to ongoing damage in the brain. In MS, relapsing and remitting episodes are unpredictable; however, peripheral inflammation may exacerbate these events. Clinical studies revealed an association between infections and relapses, which may lead to the worsening of neurological damage. A similar scenario was described in MS animal models demonstrating that peripheral inflammation recrudesced a central ongoing demyelinating lesion. In this paper, we reviewed the existing data on the inflammatory component of MS, with special attention on the effect of peripheral infections in the etiology and progression of MS and its effect on the relapsing and remitting episodes. We also analyzed data concerning the effect of peripheral inflammatory events in MS experimental animal models. This article is part of a Special Issue entitled 'Neuroinflammation in neurodegeneration and neurodysfunction'.

Accelerated axonal loss following acute CNS demyelination in mice lacking protein tyrosine phosphatase receptor type Z.

Protein tyrosine phosphatase receptor type Z (Ptprz) is widely expressed in the mammalian central nervous system and has been suggested to regulate oligodendrocyte survival and differentiation. We investigated the role of Ptprz in oligodendrocyte remyelination after acute, toxin-induced demyelination in Ptprz null mice. We found neither obvious impairment in the recruitment of oligodendrocyte precursor cells, astrocytes, or reactive microglia/macrophage to lesions nor a failure for oligodendrocyte precursor cells to differentiate and remyelinate axons at the lesions. However, we observed an unexpected increase in the number of dystrophic axons by 3 days after demyelination, followed by prominent Wallerian degeneration by 21 days in the Ptprz-deficient mice. Moreover, quantitative gait analysis revealed a deficit of locomotor behavior in the mutant mice, suggesting increased vulnerability to axonal injury. We propose that Ptprz is necessary to maintain central nervous system axonal integrity in a demyelinating environment and may be an important target of axonal protection in inflammatory demyelinating diseases, such as multiple sclerosis and periventricular leukomalacia.

CNS response to a second pro-inflammatory event depends on whether the primary demyelinating lesion is active or resolved.

Interleukin-1ß (IL-1ß) is considered to be one of the most important mediators in the pathogenesis of inflammatory diseases, particularly in neurodegenerative diseases such as multiple sclerosis (MS). MS is a chronic inflammatory disease characterized by demyelination and remyelination events, with unpredictable relapsing and remitting episodes that seldom worsen MS lesions. We proposed to study the effect of a unique component of the inflammatory process, IL-1ß, and evaluate its effect in repeated episodes, similar to the relapsing-remitting MS pathology. Using adenoviral vectors, we developed a model of focal demyelination/remyelination triggered by the chronic expression of IL-1ß. The long-term expression of IL-1ß in the striatum produced blood-brain barrier (BBB) breakdown, demyelination, microglial/macrophage activation, and neutrophil infiltration but no overt neuronal degeneration. This demyelinating process was followed by complete remyelination of the area. This simple model allows us to study demyelination and remyelination independently of the autoimmune and adaptive immune components. Re-exposure to this cytokine when the first inflammatory response was still unresolved generated a lesion with decreased neuroinflammation, demyelination, axonal injury and glial response. However, a second long-term expression of IL-1ß when the first lesion was resolved could not be differentiated from the first event. In this study, we demonstrated that the response to a second inflammatory stimulus varies depending on whether the initial lesion is still active or has been resolved. Considering that anti-inflammatory treatments have shown little improvement in MS patients, studies about the behavior of specific components of the inflammatory process should be taken into account to develop new therapeutic tools.

Notch signaling proteins HES-1 and Hey-1 bind to insulin degrading enzyme (IDE) proximal promoter and repress its transcription and activity: implications for cellular Aß metabolism.

Cerebral amyloid ß (Aß) accumulation is pathogenically associated with sporadic Alzheimer's disease (SAD). BACE-1 is involved in Aß generation while insulin-degrading enzyme (IDE) partakes in Aß proteolytic clearance. Vulnerable regions in AD brains show increased BACE-1 protein levels and enzymatic activity while the opposite occurs with IDE. Another common feature in SAD brains is Notch1 overexpression. Here we demonstrate an increase in mRNA levels of Hey-1, a Notch target gene, and a decrease of IDE transcripts in the hippocampus of SAD brains as compared to controls. Transient transfection of Notch intracellular domain (NICD) in N2aSW cells, mouse neuroblastoma cells (N2a) stably expressing human amyloid precursor protein (APP) Swedish mutation, reduce IDE mRNA levels, promoting extracellular Aß accumulation. Also, NICD, HES-1 and Hey-1 overexpression result in decreased IDE proximal promoter activity. This effect was mediated by 2 functional sites located at -379/-372 and -310-303 from the first translation start site in the -575/-19 (556 bp) fragment of IDE proximal promoter. By site-directed mutagenesis of the IDE promoter region we reverted the inhibitory effect mediated by NICD transfection suggesting that these sites are indeed responsible for the Notch-mediated inhibition of the IDE gene expression. Intracranial injection of the Notch ligand JAG-1 in Tg2576 mice, expressing the Swedish mutation in human APP, induced overexpression of HES-1 and Hey-1 and reduction of IDE mRNA levels, respectively. Our results support our theory that a Notch-dependent IDE transcriptional modulation may impact on Aß metabolism providing a functional link between Notch signaling and the amyloidogenic pathway in SAD.

Parkinson's disease and systemic inflammation.

Peripheral inflammation triggers exacerbation in the central brain's ongoing damage in several neurodegenerative diseases. Systemic inflammatory stimulus induce a general response known as sickness behaviour, indicating that a peripheral stimulus can induce the synthesis of cytokines in the brain. In Parkinson's disease (PD), inflammation was mainly associated with microglia activation that can underlie the neurodegeneration of neurons in the substantia nigra (SN). Peripheral inflammation can transform the "primed" microglia into an "active" state, which can trigger stronger responses dealing with neurodegenerative processes. Numerous evidences show that systemic inflammatory processes exacerbate ongoing neurodegeneration in PD patient and animal models. Anti-inflammatory treatment in PD patients exerts a neuroprotective effect. In the present paper, we analyse the effect of peripheral infections in the etiology and progression in PD patients and animal models, suggesting that these peripheral immune challenges can exacerbate the symptoms in the disease.

Neurogenic niche modulation by activated microglia: transforming growth factor beta increases neurogenesis in the adult dentate gyrus.

Adult neural stem cells (NSC) proliferate and differentiate depending on the composition of the cellular and molecular niche in which they are immersed. Until recently, microglial cells have been ignored as part of the neurogenic niche. We studied the dynamics of NSC proliferation and differentiation in the dentate gyrus of the hippocampus (DG) and characterized the changes of the neurogenic niche in adrenalectomized animals (ADX). At the cellular level, we found increased NSC proliferation and neurogenesis in the ADX animals. In addition, a morphologically distinct subpopulation of NSC (Nestin+/GFAP-) with increased proliferating profile was detected. Interestingly, the number of microglial cells at stages 2 and 3 of activation correlated with increased neurogenesis (r2 = 0.999) and the number of Nestin-positive cells (r2 = 0.96). At the molecular level, transforming growth factor beta (TGF-beta) mRNA levels were increased 10-fold in ADX animals. Interestingly, TGF-beta levels correlated with the amount of neurogenesis detected (r2 = 0.99) and the number of stage 2 and 3 microglial cells (r2 = 0.94). Furthermore, blockade of TGF-beta biological activity by administration of an anti-TGF-beta type II receptor antibody diminished the percentage of 5-bromo-2'-deoxyuridine (BrdU)/PSA-NCAM-positive cells in vivo. Moreover, TGF-beta was able to promote neurogenesis in NSC primary cultures. This work supports the idea that activated microglial cells are not pro- or anti-neurogenic per se, but the balance between pro- and anti-inflammatory secreted molecules influences the final effect of this activation. Importantly, we identified an anti-inflammatory cytokine, TGF-beta, with neurogenic potential in the adult brain.

Neuronal differentiation in the adult hippocampus recapitulates embryonic development.

In the adult hippocampus and olfactory bulb, neural progenitor cells generate neurons that functionally integrate into the existing circuits. To understand how neuronal differentiation occurs in the adult hippocampus, we labeled dividing progenitor cells with a retrovirus expressing green fluorescent protein and studied the morphological and functional properties of their neuronal progeny over the following weeks. During the first week neurons had an irregular shape and immature spikes and were synaptically silent. Slow GABAergic synaptic inputs first appeared during the second week, when neurons exhibited spineless dendrites and migrated into the granule cell layer. In contrast, glutamatergic afferents were detected by the fourth week in neurons displaying mature excitability and morphology. Interestingly, fast GABAergic responses were the latest to appear. It is striking that neuronal maturation in the adult hippocampus follows a precise sequence of connectivity (silent --> slow GABA --> glutamate --> fast GABA) that resembles hippocampal development. We conclude that, unlike what is observed in the olfactory bulb, the hippocampus maintains the same developmental rules for neuronal integration through adulthood.

Reversible demyelination, blood-brain barrier breakdown, and pronounced neutrophil recruitment induced by chronic IL-1 expression in the brain.

Interleukin-1beta (IL-1) expression is associated with a spectrum of neuroinflammatory processes related to chronic neurodegenerative diseases. The single-bolus microinjection of IL-1 into the central nervous system (CNS) parenchyma gives rise to delayed and localized neutrophil recruitment, transient blood-brain barrier (BBB) breakdown, but no overt damage to CNS integrity. However, acute microinjections of IL-1 do not mimic the chronic IL-1 expression, which is a feature of many CNS diseases. To investigate the response of the CNS to chronic IL-1 expression, we injected a recombinant adenovirus expressing IL-1 into the striatum. At the peak of IL-1 expression (days 8 and 14 post-injection), there was a marked recruitment of neutrophils, vasodilatation, and breakdown of the BBB. Microglia and astrocyte activation was evident during the first 14 days post-injection. At days 8 and 14, extensive demyelination was observed but the number of neurons was not affected by any treatment. Finally, at 30 days, signs of inflammation were no longer present, there was evidence of tissue reorganization, the BBB was intact, and the process of remyelination was noticeable. In summary, our data show that chronic expression of IL-1, in contrast to its acute delivery, can reversibly damage CNS integrity and implicates this cytokine or downstream components as major mediators of demyelination in chronic inflammatory and demyelinating diseases.

Spatiotemporal distribution of the insulin-like growth factor receptor in the rat olfactory bulb.

Insulin-like growth factor I (IGF-I) and its receptor (IGF-IR) are involved in growth of neurons. In the rat olfactory epithelium, we previously showed IGF-IR immunostaining in subsets of olfactory receptor neurons. We now report that IGF-IR staining was heaviest in the olfactory nerve layer of the rat olfactory bulb at embryonic days 18, and 19 and postnatal day 1, with labeling of protoglomeruli. In the adult, only a few glomeruli were IGF-IR-positive, some of which were unusually small and strongly labeled. Some IGF-IR-positive fibers penetrated deeper into the external plexiform layer, even in adults. In developing tissues, IGF-IR staining co-localized with that for olfactory marker protein and growth associated protein GAP-43, but to a lesser extent with synaptophysin. In the adult, IGF-IR-positive fibers were compartmentalized within glomeruli. IGF-I may play a role in glomerular synaptogenesis and/or plasticity, possibly contributing to development of coding patterns for odor detection or identification.

Standardization of fixation, processing and staining methods for the central nervous system of vertebrates

This paper reports the standardization of methods used for processing and embedding various vertebrate brains of different size in paraffin. Other technical details developed for avoiding frequent difficulties arising during laboratory routine are also reported. Some modifications of the Nissl and Kl³ver-Barrera staining methods are proposed. These modifications include: 1) a Nissl stain solution with a rapid and efficient action with easier differentiation; 2) the use of a cheap microwave oven for the Kl³ver-Barrera stain. These procedures have the advantage of permitting Nissl and Kl³ver-Barrera staining of nervous tissue in about five and fifteen minutes respectively. The proposed procedures have been tested in brains obtained from fish, amphibians, reptiles and mammals of different body sizes. They are the result of our long experience in preparing slides for comparative studies. Serial sections of excellent quality were regularly obtained in all the specimens studied. These standardized methods, being simple and quick, are recommended for routine use in neurobiological laboratories.(AU)

Standardization of fixation, processing and staining methods for the central nervous system of vertebrates

This paper reports the standardization of methods used for processing and embedding various vertebrate brains of different size in paraffin. Other technical details developed for avoiding frequent difficulties arising during laboratory routine are also reported. Some modifications of the Nissl and Klüver-Barrera staining methods are proposed. These modifications include: 1) a Nissl stain solution with a rapid and efficient action with easier differentiation; 2) the use of a cheap microwave oven for the Klüver-Barrera stain. These procedures have the advantage of permitting Nissl and Klüver-Barrera staining of nervous tissue in about five and fifteen minutes respectively. The proposed procedures have been tested in brains obtained from fish, amphibians, reptiles and mammals of different body sizes. They are the result of our long experience in preparing slides for comparative studies. Serial sections of excellent quality were regularly obtained in all the specimens studied. These standardized methods, being simple and quick, are recommended for routine use in neurobiological laboratories.