Santiago Rivera

Resuscitation of hypoxic piglets with 100% O2 increases pulmonary metalloproteinases and IL-8.

We hypothesized that resuscitation with 100% O2 compared with 21% O2 is detrimental to pulmonary tissue. The pulmonary injury was assessed by matrix metalloproteinase (MMP) activity, oxidative stress, IL-8, and histology 2.5 h after resuscitation from a hypoxic state. In pulmonary tissue extracts, MMP activity was analyzed by broad matrix–degrading capacity (total MMP) and zymography. MMP-2 mRNA expression was evaluated by quantitative real-time PCR. Total endogenous antioxidant capacity was measured by the oxygen radical absorbance capacity (ORAC) assay, and IL-8 was analyzed by ELISA technique. In bronchoalveolar lavage (BAL) fluid, MMPs were analyzed by zymography. In pulmonary tissue, pro- and active MMP-2 levels were increased in piglets that were resuscitated with 100% O2 compared with 21% O2. Pro–MMP-9, total MMP activity, and MMP-2 mRNA levels were significantly increased in resuscitated piglets compared with baseline. Net gelatinolytic activity increased in submucosa and blood vessels after 100% O2 and only in the blood vessels after 21% O2. Compared with baseline, ORAC values were considerably lowered in the resuscitated piglets and significantly reduced in the 100% O2 versus 21% O2 group. In BAL fluid, both pro–MMP-9 and pro–MMP-2 increased 2-fold in the 100% O2 group compared with 21% O2. Moreover, IL-8 concentration increased significantly in piglets that were resuscitated with 100% O2 compared with 21% O2, suggesting a marked proinflammatory response in the pulmonary tissue. Altogether, these data strongly suggest that caution must be taken when applying pure O2 to the newborn infant.

Temporal gene profiling of the 5XFAD transgenic mouse model highlights the importance of microglial activation in Alzheimer’s disease

BackgroundThe 5XFAD early onset mouse model of Alzheimer’s disease (AD) is gaining momentum. Behavioral, electrophysiological and anatomical studies have identified age-dependent alterations that can be reminiscent of human AD. However, transcriptional changes during disease progression have not yet been investigated. To this end, we carried out a transcriptomic analysis on RNAs from the neocortex and the hippocampus of 5XFAD female mice at the ages of one, four, six and nine months (M1, M4, M6, M9).ResultsOur results show a clear shift in gene expression patterns between M1 and M4. At M1, 5XFAD animals exhibit region-specific variations in gene expression patterns whereas M4 to M9 mice share a larger proportion of differentially expressed genes (DEGs) that are common to both regions. Analysis of DEGs from M4 to M9 underlines the predominance of inflammatory and immune processes in this AD mouse model. The rise in inflammation, sustained by the overexpression of genes from the complement and integrin families, is accompanied by an increased expression of transcripts involved in the NADPH oxidase complex, phagocytic processes and IFN-γ related pathways.ConclusionsOverall, our data suggest that, from M4 to M9, sustained microglial activation becomes the predominant feature and point out that both detrimental and neuroprotective mechanisms appear to be at play in this model. Furthermore, our study identifies a number of genes already known to be altered in human AD, thus confirming the use of the 5XFAD strain as a valid model for understanding AD pathogenesis and for screening potential therapeutic molecules.

The potential role of metalloproteinases in neurogenesis in the gerbil hippocampus following global forebrain ischemia.

Background Matrix metalloproteinases (MMPs) have recently been considered to be involved in the neurogenic response of adult neural stem/progenitor cells. However, there is a lack of information showing direct association between the activation of MMPs and the development of neuronal progenitor cells involving proliferation and/or further differentiation in vulnerable (Cornus Ammoni-CA1) and resistant (dentate gyrus-DG) to ischemic injury areas of the brain hippocampus. Principal Findings We showed that dynamics of MMPs activation in the dentate gyrus correlated closely with the rate of proliferation and differentiation of progenitor cells into mature neurons. In contrast, in the damaged CA1 pyramidal cells layer, despite the fact that some proliferating cells exhibited antigen specific characteristic of newborn neuronal cells, these did not attain maturity. This coincides with the low, near control-level, activity of MMPs. The above results are supported by our in vitro study showing that MMP inhibitors interfered with both the proliferation and differentiation of the human neural stem cell line derived from umbilical cord blood (HUCB-NSCs) toward the neuronal lineage. Conclusion Taken together, the spatial and temporal profiles of MMPs activity suggest that these proteinases could be an important component in neurogenesis-associated processes in post-ischemic brain hippocampus.

Early administration of RS 67333, a specific 5-HT4 receptor agonist, prevents amyloidogenesis and behavioral deficits in the 5XFAD mouse model of Alzheimer’s disease

Amyloid β (Aβ) accumulation is considered the main culprit in the pathogenesis of Alzheimer’s disease (AD). Recent studies suggest that decreasing Aβ production at very early stages of AD could be a promising strategy to slow down disease progression. Serotonin 5-HT4 receptor activation stimulates α-cleavage of the amyloid precursor protein (APP), leading to the release of the soluble and neurotrophic sAPPα fragment and thus precluding Aβ formation. Using the 5XFAD mouse model of AD that shows accelerated Aβ deposition, we investigated the effect of chronic treatments (treatment onset at different ages and different durations) with the 5-HT4 receptor agonist RS 67333 during the asymptomatic phase of the disease. Chronic administration of RS 67333 decreased concomitantly the number of amyloid plaques and the level of Aβ species. Reduction of Aβ levels was accompanied by a striking decrease in hippocampal astrogliosis and microgliosis. RS 67333 also transiently increased sAPPα concentration in the cerebrospinal fluid and brain. Moreover, a specific 5-HT4 receptor antagonist (RS 39604) prevented the RS 67333-mediated reduction of the amyloid pathology. Finally, the novel object recognition test deficits of 5XFAD mice were reversed by chronic treatment with RS 67333. Collectively, these results strongly highlight this 5-HT4 receptor agonist as a promising disease modifying-agent for AD.

Trafficking and secretion of matrix metalloproteinase-2 in olfactory ensheathing glial cells: A role in cell migration?

Olfactory ensheathing cells (OECs) are unique glia found only in the olfactory system. They retain exceptional plasticity and support olfactory neurogenesis and retargeting across the PNS:CNS boundary in the olfactory system. OECs have been shown to improve functional outcome when transplanted into rodents with spinal cord injury. The growth‐promoting properties of implanted OECs encompass their ability to migrate through the scar tissue and render it more permissive for axonal outgrowth, but the underlying molecular mechanisms remain poorly understood. OECs appear to regulate molecules of the extracellular matrix (ECM) that inhibit axonal growth. Among the proteins that have the potential to promote cell migration, axonal regeneration and remodeling of the ECM are matrix metalloproteinases (MMPs), a family of endopeptidases that cleave matrix, soluble, and membrane‐bound proteins and that are regulated by their endogenous inhibitors, the tissue inhibitors of MMPs (TIMPs). Little is known about MMP/TIMP trafficking, secretion, and role in OECs. Using a combination of cell biology, biochemistry, pharmacology, and imaging techniques, we show that MMP‐2 and MMP‐9 are expressed and proteolytically active in the olfactory epithelium and in particular in the OECs of the lamina propria. These proteinases and regulatory proteins such as MT1‐MMP and TIMP‐2 are expressed in cultured OECs. MMPs exhibit nuclear localization and vesicular trafficking and secretion, with distribution along microtubules and microfilaments and co‐localization with the molecular motor protein kinesin. Finally, we show that MMPs are involved in migration of OECs in vitro on different ECM substrates.

Evidence for early cognitive impairment related to frontal cortex in the 5XFAD mouse model of Alzheimer's disease.

The frontal cortex is a brain structure that plays an important role in cognition and is known to be affected in Alzheimers disease (AD) in humans. Over the past years, transgenic mouse models have been generated to recapitulate the main features of this disease, including cognitive impairments. This study investigates frontal cortex dependent learning abilities in one of the most early-onset transgenic murine model of AD, the 5XFAD mice. We compared frontal performance of 2-, 4-, and 6-month-old 5XFAD mice with their wild-type littermates using a newly developed automated device, the olfactory H-maze, in which mice have to discover three different rules consecutively according to the delayed reaction paradigm. We report early cognitive deficits related to frontal cortex appearing in 4-month-old 5XFAD mice before hippocampal-dependent learning and memory impairment, in relation with neuropathologic processes such as strong gliosis and emerging amyloid plaques. The present results demonstrate that the olfactory H-maze is a very sensitive and simple experimental paradigm that allows assessment of frontal functions in transgenic mice and should be useful to test pre-clinical therapeutic approaches to alter the course of AD.

Area-specific alterations of synaptic plasticity in the 5XFAD mouse model of Alzheimer's disease: dissociation between somatosensory cortex and hippocampus.

Transgenic mouse models of Alzheimer’s disease (AD) that overproduce the amyloid beta peptide (Aβ) have highlighted impairments of hippocampal long-term synaptic plasticity associated with the progression of the disease. Here we examined whether the characteristics of one of the hallmarks of AD, i.e. Aβ deposition, in both the somatosensory cortex and the hippocampus, correlated with specific losses of synaptic plasticity in these areas. For this, we evaluated the occurrence of long-term potentiation (LTP) in the cortex and the hippocampus of 6-month old 5xFAD transgenic mice that exhibited massive Aβ deposition in both regions but with different features: in cortical areas a majority of Aβ deposits comprised a dense core surrounded by a diffuse corona while such kind of Aβ deposition was less frequently observed in the hippocampus. In order to simultaneously monitor synaptic changes in both areas, we developed a method based on the use of Multi-Electrode Arrays (MEA). When compared with wild-type (WT) mice, basal transmission was significantly reduced in both areas in 5xFAD mice, while short-term synaptic plasticity was unaffected. The induction of long-term changes of synaptic transmission by different protocols revealed that in 5xFAD mice, LTP in the layer 5 of the somatosensory cortex was more severely impaired than LTP triggered in the CA1 area of the hippocampus. We conclude that cortical plasticity is deficient in the 5xFAD model and that this deficit could be correlated with the proportion of diffuse plaques in 5xFAD mice.

Role of matrix metalloproteinases in migration and neurotrophic properties of nasal olfactory stem and ensheathing cells.

Adult olfactory ectomesenchymal stem cells (OE-MSCs) and olfactory ensheathing cells (OECs), both from the nasal olfactory lamina propria, display robust regenerative properties when transplanted into the nervous system, but the mechanisms supporting such therapeutic effects remain unknown. Matrix metalloproteinases (MMPs) are an important family of proteinases contributing to cell motility and axonal outgrowth across the extracellular matrix (ECM) in physiological and pathological conditions. In this study, we have characterized for the first time in nasal human OE-MSCs the expression profile of some MMPs currently associated with cell migration and invasiveness. We demonstrate different patterns of expression for MMP-1, MMP-2, MMP-9, and MT1-MMP upon cell migration when compared with nonmigrating cells. Our results establish a correspondence between the localization of these proteinases in the migration front with the ability of cells to migrate. Using various modulators of MMP activity, we also show that at least MMP-2, MMP-9, and MT1-MMP contribute to OE-MSC migration in an in vitro 3D test. Furthermore, we demonstrate under the same conditions of culture used for in vivo transplantation that OE-MSCs and OECs secrete neurotrophic factors that promote neurite outgrowth of cortical and dorsal root ganglia (DRG) neurons, as well as axo-dendritic differentiation of cortical neurons. These effects were abolished by the depletion of MMP-2 and MMP-9 from the culture conditioned media. Altogether, our results provide the first evidence that MMPs may contribute to the therapeutic features of OE-MSCs and OECs through the control of their motility and/or their neurotrophic properties. Our data provide new insight into the mechanisms of neuroregeneration and will contribute to optimization of cell therapy strategies.

Differential spatio-temporal regulation of MMPs in the 5xFAD mouse model of Alzheimer’s disease: evidence for a pro-amyloidogenic role of MT1-MMP

Matrix metalloproteinases (MMPs) are pleiotropic endopeptidases involved in a variety of neurodegenerative/neuroinflammatory processes through their interactions with a large number of substrates. Among those, the amyloid precursor protein (APP) and the beta amyloid peptide (Aβ) are largely associated with the development of Alzheimer’s disease (AD). However, the regulation and potential contribution of MMPs to AD remains unclear. In this study, we investigated the evolution of the expression of MMP-2, MMP-9, and membrane-type 1-MMP (MT1-MMP) in the hippocampus at different stages of the pathology (asymptomatic, prodromal-like and symptomatic) in the 5xFAD transgenic mouse AD model. In parallel we also followed the expression of functionally associated factors. Overall, the expression of MMP-2, MMP-9, and MT1-MMP was upregulated concomitantly with the tissue inhibitor of MMPs-1 (TIMP-1) and several markers of inflammatory/glial response. The three MMPs exhibited age- and cell-dependent upregulation of their expression, with MMP-2 and MMP-9 being primarily located to astrocytes, and MT1-MMP to neurons. MMP-9 and MT1-MMP were also prominently present in amyloid plaques. The levels of active MT1-MMP were highly upregulated in membrane-enriched fractions of hippocampus at 6 months of age (symptomatic phase), when the levels of APP, its metabolites APP C-terminal fragments (CTFs), and Aβ trimers were the highest. Overexpression of MT1-MMP in HEK cells carrying the human APP Swedish mutation (HEKswe) strongly increased β-secretase derived C-terminal APP fragment (C99) and Aβ levels, whereas MMP-2 overexpression nearly abolished Aβ production without affecting C99. Our data consolidate the emerging idea of a regulatory interplay between MMPs and the APP/Aβ system, and demonstrate for the first time the pro-amyloidogenic features of MT1-MMP. Further investigation will be justified to evaluate this MMP as a novel potential therapeutic target in AD.

Onset of hippocampus-dependent memory impairments in 5XFAD transgenic mouse model of Alzheimer's disease.

The 5XFAD mice are an early‐onset transgenic model of Alzheimers disease (AD) in which amyloid plaques are first observed between two and four months of age in the cortical layer five and in the subiculum of the hippocampal formation. Although cognitive alterations have been described in these mice, there are no studies that focused on the onset of hippocampus‐dependent memory deficits, which are a hallmark of the prodromal stage of AD. To identify when the first learning and memory impairments appear, 5XFAD mice of two, four, and six months of age were compared with their respective wild‐type littermates using the olfactory tubing maze, which is a very sensitive hippocampal‐dependent task. Deficits in learning and memory started at four months with a substantial increase at six months of age while no olfactory impairments were observed. The volumetric study using magnetic resonance imaging of the whole brain and specific areas (olfactory bulb, striatum, and hippocampus) did not reveal neuro‐anatomical difference. Slight memory deficits appeared at 4 months of age in correlation with an increased astrogliosis and amyloid plaque formation. This early impairment in learning and memory related to the hippocampal dysfunction is particularly suited to assess preclinical therapeutic strategies aiming to delay or suppress the onset of AD.