The Helico Maze Detects Early Impairment of Reference Memory at Three Months of Age in the 5XFAD Mouse Model of Alzheimer's Disease.

BACKGROUND: The 5XFAD model of Alzheimer's disease (AD) bearing five familial mutations of Alzheimer's disease on human APP and PSEN1 transgenes shows deposits of amyloid-ß peptide (Aß) as early as 2 months, while deficits in long-term memory can be detected at 4 months using the highly sensitive olfactory-dependent tests that we previously reported. OBJECTIVE: Given that detecting early dysfunctions in AD prior to overt pathology is of major interest in the field, we sought to detect memory deficits at earlier stages of the disease in 3-month-old male 5XFAD mice. METHODS: To this end, we used the Helico Maze, a behavioral task that was recently developed and patented. This device allows deeper analysis of learning and subcategories of hippocampal-dependent long-term memory using olfactory cues. RESULTS: Eight male 5XFAD and 6 male wild-type (WT: C57Bl6 background) mice of 3 months of age were tested in the Helico Maze. The results demonstrated, for the first time, a starting deficit of pure reference long-term memory. Interestingly, memory impairment was clearly correlated with Aß deposits in the hippocampus. While we also found significant differences in astrogliosis between 5XFAD and WT mice, this was not correlated with memory abilities. CONCLUSION: Our results underline the efficiency of this new olfactory-dependent behavioral task, which is easy to use, with a small cohort of mice. Using the Helico Maze may open new avenues to validate the efficacy of treatments that target early events related to the amyloid-dependent pathway of the disease and AD progression.

APOE4 drives inflammation in human astrocytes via TAGLN3 repression and NF-κB activation.

Apolipoprotein E4 (APOEε4) is the major allelic risk factor for late-onset sporadic Alzheimer's disease (sAD). Inflammation is increasingly considered as critical in sAD initiation and progression. Identifying brain molecular mechanisms that could bridge these two risk factors remain unelucidated. Leveraging induced pluripotent stem cell (iPSC)-based strategies, we demonstrate that APOE controls inflammation in human astrocytes by regulating Transgelin 3 (TAGLN3) expression and, ultimately, nuclear factor κB (NF-κB) activation. We uncover that APOE4 specifically downregulates TAGLN3, involving histone deacetylases activity, which results in low-grade chronic inflammation and hyperactivated inflammatory responses. We show that APOE4 exerts a dominant negative effect to prime astrocytes toward a pro-inflammatory state that is pharmacologically reversible by TAGLN3 supplementation. We further confirm that TAGLN3 is downregulated in the brain of patients with sAD. Our findings highlight the APOE-TAGLN3-NF-κB axis regulating neuroinflammation in human astrocytes and reveal TAGLN3 as a molecular target to modulate neuroinflammation, as well as a potential biomarker for AD.

MT5-MMP promotes neuroinflammation, neuronal excitability and Aß production in primary neuron/astrocyte cultures from the 5xFAD mouse model of Alzheimer's disease.

BACKGROUND: Membrane-type matrix metalloproteinase 5 (MT5-MMP) deficiency in the 5xFAD mouse model of Alzheimer's disease (AD) reduces brain neuroinflammation and amyloidosis, and prevents deficits in synaptic activity and cognition in prodromal stages of the disease. In addition, MT5-MMP deficiency prevents interleukin-1 beta (IL-1ß)-mediated inflammation in the peripheral nervous system. In this context, we hypothesized that the MT5-MMP/IL-1ß tandem could regulate nascent AD pathogenic events in developing neural cells shortly after the onset of transgene activation. METHODS: To test this hypothesis, we used 11-14 day in vitro primary cortical cultures from wild type, MT5-MMP-/-, 5xFAD and 5xFAD/MT5-MMP-/- mice, and evaluated the impact of MT5-MMP deficiency and IL-1ß treatment for 24 h, by performing whole cell patch-clamp recordings, RT-qPCR, western blot, gel zymography, ELISA, immunocytochemistry and adeno-associated virus (AAV)-mediated transduction. RESULTS: 5xFAD cells showed higher levels of MT5-MMP than wild type, concomitant with higher basal levels of inflammatory mediators. Moreover, MT5-MMP-deficient cultures had strong decrease of the inflammatory response to IL-1ß, as well as decreased stability of recombinant IL-1ß. The levels of amyloid beta peptide (Aß) were similar in 5xFAD and wild-type cultures, and IL-1ß treatment did not affect Aß levels. Instead, the absence of MT5-MMP significantly reduced Aß by more than 40% while sparing APP metabolism, suggesting altogether no functional crosstalk between IL-1ß and APP/Aß, as well as independent control of their levels by MT5-MMP. The lack of MT5-MMP strongly downregulated the AAV-induced neuronal accumulation of the C-terminal APP fragment, C99, and subsequently that of Aß. Finally, MT5-MMP deficiency prevented basal hyperexcitability observed in 5xFAD neurons, but not hyperexcitability induced by IL-1ß treatment. CONCLUSIONS: Neuroinflammation and hyperexcitability precede Aß accumulation in developing neural cells with nascent expression of AD transgenes. MT5-MMP deletion is able to tune down basal neuronal inflammation and hyperexcitability, as well as APP/Aß metabolism. In addition, MT5-MMP deficiency prevents IL-1ß-mediated effects in brain cells, except hyperexcitability. Overall, this work reinforces the idea that MT5-MMP is at the crossroads of pathogenic AD pathways that are already incipiently activated in developing neural cells, and that targeting MT5-MMP opens interesting therapeutic prospects.

A Marine λ-Oligocarrageenan Inhibits Migratory and Invasive Ability of MDA-MB-231 Human Breast Cancer Cells through Actions on Heparanase Metabolism and MMP-14/MMP-2 Axis.

Sugar-based molecules such as heparins or natural heparan sulfate polysaccharides have been developed and widely studied for controlling heparanase (HPSE) enzymatic activity, a key player in extracellular matrix remodelling during cancer pathogenesis. However, non-enzymatic functions of HPSE have also been described in tumour mechanisms. Given their versatile properties, we hypothesized that sugar-based inhibitors may interfere with enzymatic but also non-enzymatic HPSE activities. In this work, we assessed the effects of an original marine λ-carrageenan derived oligosaccharide (λ-CO) we previously described, along with those of its native counterpart and heparins, on cell viability, proliferation, migration, and invasion of MDA-MB-231 breast cancer cells but also of sh-MDA-MB-231 cells, in which the expression of HPSE was selectively downregulated. We observed no cytotoxic and no anti-proliferative effects of our compounds but surprisingly λ-CO was the most efficient to reduce cell migration and invasion compared with heparins, and in a HPSE-dependent manner. We provided evidence that λ-CO tightly controlled a HPSE/MMP-14/MMP-2 axis, leading to reduced MMP-2 activity. Altogether, this study highlights λ-CO as a potent HPSE "modulator" capable of reducing not only the enzymatic activity of HPSE but also the functions controlled by the HPSE levels.

MT5-MMP controls APP and ß-CTF/C99 metabolism through proteolytic-dependent and -independent mechanisms relevant for Alzheimer's disease.

We previously discovered the implication of membrane-type 5-matrix metalloproteinase (MT5-MMP) in Alzheimer's disease (AD) pathogenesis. Here, we shed new light on pathogenic mechanisms by which MT5-MMP controls the processing of amyloid precursor protein (APP) and the fate of amyloid beta peptide (Aß) as well as its precursor C99, and C83. We found in human embryonic kidney cells (HEK) carrying the APP Swedish familial mutation (HEKswe) that deleting the C-terminal non-catalytic domains of MT5-MMP hampered its ability to process APP and release the soluble 95 kDa form (sAPP95). Catalytically inactive MT5-MMP variants increased the levels of Aß and promoted APP/C99 sorting in the endolysosomal system, likely through interactions of the proteinase C-terminal portion with C99. Most interestingly, the deletion of the C-terminal domain of MT5-MMP caused a strong degradation of C99 by the proteasome and prevented Aß accumulation. These discoveries reveal new control of MT5-MMP over APP by proteolytic and non-proteolytic mechanisms driven by the C-terminal domains of the proteinase. The targeting of these non-catalytic domains of MT5-MMP could, therefore, provide new insights into the therapeutic regulation of APP-related pathology in AD.

Matrix Metalloproteinases as New Targets in Alzheimer's Disease: Opportunities and Challenges.

Although matrix metalloproteinases (MMPs) are implicated in the regulation of numerous physiological processes, evidence of their pathological roles have also been obtained in the last decades, making MMPs attractive therapeutic targets for several diseases. Recent discoveries of their involvement in central nervous system (CNS) disorders, and in particular in Alzheimer's disease (AD), have paved the way to consider MMP modulators as promising therapeutic strategies. Over the past few decades, diverse approaches have been undertaken in the design of therapeutic agents targeting MMPs for various purposes, leading, more recently, to encouraging developments. In this article, we will present recent examples of inhibitors ranging from small molecules and peptidomimetics to biologics. We will also discuss the scientific knowledge that has led to the development of emerging tools and techniques to overcome the challenges of selective MMP inhibition.

Donecopride, a Swiss army knife with potential against Alzheimer's disease.

BACKGROUND AND PURPOSE: We recently identified donecopride as a pleiotropic compound able to inhibit AChE and to activate 5-HT4 receptors. Here, we have assessed the potential therapeutic effects of donecopride in treating Alzheimer's disease (AD). EXPERIMENTAL APPROACH: We used two in vivo animal models of AD, transgenic 5XFAD mice and mice exposed to soluble amyloid-ß peptides and, in vitro, primary cultures of rat hippocampal neurons. Pro-cognitive and anti-amnesic effects were evaluated with novel object recognition, Y-maze, and Morris water maze tests. Amyloid load in mouse brain was measured ex vivo and effects of soluble amyloid-ß peptides on neuronal survival and neurite formation determined in vitro. KEY RESULTS: In vivo, chronic (3 months) administration of donecopride displayed potent anti-amnesic properties in the two mouse models of AD, preserving learning capacities, including working and long-term spatial memories. These behavioural effects were accompanied by decreased amyloid aggregation in the brain of 5XFAD mice and, in cultures of rat hippocampal neurons, reduced tau hyperphosphorylation. In vitro, donecopride increased survival in neuronal cultures exposed to soluble amyloid-ß peptides, improved the neurite network and provided neurotrophic benefits, expressed as the formation of new synapses. CONCLUSIONS AND IMPLICATIONS: Donecopride acts like a Swiss army knife, exhibiting a range of sustainable symptomatic therapeutic effects and potential disease-modifying effects in models of AD. Clinical trials with this promising drug candidate will soon be undertaken to confirm its therapeutic potential in humans.

Emerging Alternative Proteinases in APP Metabolism and Alzheimer's Disease Pathogenesis: A Focus on MT1-MMP and MT5-MMP.

Processing of amyloid beta precursor protein (APP) into amyloid-beta peptide (Aß) by ß-secretase and γ-secretase complex is at the heart of the pathogenesis of Alzheimer's disease (AD). Targeting this proteolytic pathway effectively reduces/prevents pathology and cognitive decline in preclinical experimental models of the disease, but therapeutic strategies based on secretase activity modifying drugs have so far failed in clinical trials. Although this may raise some doubts on the relevance of ß- and γ-secretases as targets, new APP-cleaving enzymes, including meprin-ß, legumain (δ-secretase), rhomboid-like protein-4 (RHBDL4), caspases and membrane-type matrix metalloproteinases (MT-MMPs/η-secretases) have confirmed that APP processing remains a solid mechanism in AD pathophysiology. This review will discuss recent findings on the roles of all these proteinases in the nervous system, and in particular on the roles of MT-MMPs, which are at the crossroads of pathological events involving not only amyloidogenesis, but also inflammation and synaptic dysfunctions. Assessing the potential of these emerging proteinases in the Alzheimer's field opens up new research prospects to improve our knowledge of fundamental mechanisms of the disease and help us establish new therapeutic strategies.

Long-Term Pantethine Treatment Counteracts Pathologic Gene Dysregulation and Decreases Alzheimer's Disease Pathogenesis in a Transgenic Mouse Model.

The low-molecular weight thiol pantethine, known as a hypolipidemic and hypocholesterolemic agent, is the major precursor of co-enzyme A. We have previously shown that pantethine treatment reduces amyloid-ß (Aß)-induced IL-1ß release and alleviates pathological metabolic changes in primary astrocyte cultures. These properties of pantethine prompted us to investigate its potential benefits in vivo in the 5XFAD (Tg) mouse model of Alzheimer's disease (AD).1.5-month-old Tg and wild-type (WT) male mice were submitted to intraperitoneal administration of pantethine or saline control solution for 5.5 months. The effects of such treatments were investigated by performing behavioral tests and evaluating astrogliosis, microgliosis, Αß deposition, and whole genome expression arrays, using RNAs extracted from the mice hippocampi. We observed that long-term pantethine treatment significantly reduced glial reactivity and Αß deposition, and abrogated behavioral alteration in Tg mice. Moreover, the transcriptomic profiles revealed that after pantethine treatment, the expression of genes differentially expressed in Tg mice, and in particular those known to be related to AD, were significantly alleviated. Most of the genes overexpressed in Tg compared to WT were involved in inflammation, complement activation, and phagocytosis and were found repressed upon pantethine treatment. In contrast, pantethine restored the expression of a significant number of genes involved in the regulation of Αß processing and synaptic activities, which were downregulated in Tg mice. Altogether, our data support a beneficial role for long-term pantethine treatment in preserving CNS crucial functions altered by Aß pathogenesis in Tg mice and highlight the potential efficiency of pantethine to alleviate AD pathology.

Metalloproteinases and their tissue inhibitors in Alzheimer's disease and other neurodegenerative disorders.

As life expectancy increases worldwide, age-related neurodegenerative diseases will increase in parallel. The lack of effective treatment strategies may soon lead to an unprecedented health, social and economic crisis. Any attempt to halt the progression of these diseases requires a thorough knowledge of the pathophysiological mechanisms involved to facilitate the identification of new targets and the application of innovative therapeutic strategies. The metzincin superfamily of metalloproteinases includes matrix metalloproteinases (MMP), a disintegrin and metalloproteinase (ADAM) and ADAM with thrombospondin motifs (ADAMTS). These multigenic and multifunctional proteinase families regulate the functions of an increasing number of signalling and scaffolding molecules involved in neuroinflammation, blood-brain barrier disruption, protein misfolding, synaptic dysfunction or neuronal death. Metalloproteinases and their physiological inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), are therefore, at the crossroads of molecular and cellular mechanisms that support neurodegenerative processes, and emerge as potential new therapeutic targets. We provide an overview of current knowledge on the role and regulation of metalloproteinases and TIMPs in four major neurodegenerative diseases: Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and Huntington's disease.

Proamyloidogenic effects of membrane type 1 matrix metalloproteinase involve MMP-2 and BACE-1 activities, and the modulation of APP trafficking.

We previously demonstrated that membrane type 1 (MT1) matrix metalloproteinase (MMP) was up-regulated in the hippocampus of the model of transgenic mice bearing 5 familial mutations on human amyloid precursor protein (APP) and presenilin 1 of Alzheimer disease (AD), and that the proteinase increased the levels of amyloid ß peptide (Aß) and its APP C-terminal fragment of 99 aa in a heterologous cell system. Here we provide further evidence that MT1-MMP interacts with APP and promotes amyloidogenesis in a proteolytic-dependent manner in Swedish APP-expressing human embryonic kidney 293 (HEKswe) cells. MT1-MMP-mediated processing of APP releases a soluble APP fragment, sAPP95. This process partly requires the activation of endogenous MMP-2 but is independent of ß-site APP cleaving enzyme 1 (BACE-1) or α-secretase activities. In contrast, MT1-MMP-mediated increase of Aß levels involved BACE-1 activity and was inhibited by tissue inhibitor of MMP-2, a natural inhibitor of both MT1-MMP and MMP-2. Interestingly, near abolishment of basal Aß production upon BACE-1 inhibition was rescued by MT1-MMP, indicating that the latter could mimic ß-secretase-like activity. Moreover, MT1-MMP promoted APP/Aß localization in endosomes, where Aß production mainly occurs. These data unveil new mechanistic insights to support the proamyloidogenic role of MT1-MMP based on APP processing and trafficking, and reinforce the idea that this proteinase may become a new potential therapeutic target in AD.-Paumier, J.-M., Py, N. A., González, L. G., Bernard, A., Stephan, D., Louis, L., Checler, F., Khrestchatisky, M., Baranger, K., Rivera, S. Proamyloidogenic effects of membrane type 1 matrix metalloproteinase involve MMP-2 and BACE-1 activities, and the modulation of APP trafficking.

Correction: Metabolic changes and inflammation in cultured astrocytes from the 5xFAD mouse model of Alzheimer's disease: Alleviation by pantethine.

[This corrects the article DOI: 10.1371/journal.pone.0175369.].

Vitamin D Improves Neurogenesis and Cognition in a Mouse Model of Alzheimer's Disease.

The impairment of hippocampal neurogenesis at the early stages of Alzheimer's disease (AD) is believed to support early cognitive decline. Converging studies sustain the idea that vitamin D might be linked to the pathophysiology of AD and to hippocampal neurogenesis. Nothing being known about the effects of vitamin D on hippocampal neurogenesis in AD, we assessed them in a mouse model of AD. In a previous study, we observed that dietary vitamin D supplementation in female AD-like mice reduced cognitive decline only when delivered during the symptomatic phase. With these data in hand, we wondered whether the consequences of vitamin D administration on hippocampal neurogenesis are stage-dependent. Male wild-type and transgenic AD-like mice (5XFAD model) were fed with a diet containing either no vitamin D (0VD) or a normal dose of vitamin D (NVD) or a high dose of vitamin D (HVD), from month 1 to month 6 (preventive arm) or from month 4 to month 9 (curative arm). Working memory was assessed using the Y-maze, while amyloid burden, astrocytosis, and neurogenesis were quantified using immunohistochemistry. In parallel, the effects of vitamin D on proliferation and differentiation were assayed on primary cultures of murine neural progenitor cells. Improved working memory and neurogenesis were observed when high vitamin D supplementation was administered during the early phases of the disease, while a normal dose of vitamin D increased neurogenesis during the late phases. Conversely, an early hypovitaminosis D increased the number of amyloid plaques in AD mice while a late hypovitaminosis D impaired neurogenesis in AD and WT mice. The observed in vivo vitamin D-associated increased neurogenesis was partially substantiated by an augmented in vitro proliferation but not an increased differentiation of neural progenitors into neurons. Finally, a sexual dimorphism was observed. Vitamin D supplementation improved the working memory of males and females, when delivered during the pre-symptomatic and symptomatic phases, respectively. Our study establishes that (i) neurogenesis is improved by vitamin D in a male mouse model of AD, in a time-dependent manner, and (ii) cognition is enhanced in a gender-associated way. Additional pre-clinical studies are required to further understand the gender- and time-specific mechanisms of action of vitamin D in AD. This may lead to an adaptation of vitamin D supplementation in relation to patient's gender and age as well as to the stage of the disease.

Chronic treatments with a 5-HT4 receptor agonist decrease amyloid pathology in the entorhinal cortex and learning and memory deficits in the 5xFAD mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the main cause of dementia and a major health issue worldwide. The complexity of the pathology continues to challenge its comprehension and the implementation of effective treatments. In the last decade, a number of possible targets of intervention have been pointed out, among which the stimulation of 5-HT4 receptors (5-HT4Rs) seems very promising. 5-HT4R agonists exert pro-cognitive effects, inhibit amyloid-ß peptide (Aß) production and therefore directly and positively impact AD progression. In the present work, we investigated the effects of RS 67333, a partial 5-HT4R agonist, after chronic administration in the 5xFAD mouse model of AD. 5xFAD male mice and their wild type (WT) male littermates received either RS 67333 or vehicle solution i.p., twice a week, for 2 or 4 months. Cognitive performance was evaluated in a hippocampal-dependent behavioral task, the olfactory tubing maze (OTM). Mice were then sacrificed to evaluate the metabolism of the amyloid precursor protein (APP), amyloidosis and neuroinflammatory processes. No beneficial effects of RS 67333 were observed in 5xFAD mice after 2 months of treatment, while 5xFAD mice treated for 4 months showed better cognitive abilities compared to vehicle-treated 5xFAD mice. The beneficial effects of RS 67333 on learning and memory correlated with the decrease in both amyloid plaque load and neuroinflammation, more specifically in the entorhinal cortex. The most significant improvements in learning and memory and reduction of pathology stigmata were observed after the 4-month administration of RS 67333, demonstrating that treatment duration is important to alleviate amyloidosis and glial reactivity, particularly in the entorhinal cortex. These results confirm the 5-HT4R as a promising target for AD pathogenesis and highlight the need for further investigations to characterize fully the underlying mechanisms of action.

From Blood to Lesioned Brain: An In Vitro Study on Migration Mechanisms of Human Nasal Olfactory Stem Cells.

Stem cell-based therapies critically rely on selective cell migration toward pathological or injured areas. We previously demonstrated that human olfactory ectomesenchymal stem cells (OE-MSCs), derived from an adult olfactory lamina propria, migrate specifically toward an injured mouse hippocampus after transplantation in the cerebrospinal fluid and promote functional recoveries. However, the mechanisms controlling their recruitment and homing remain elusive. Using an in vitro model of blood-brain barrier (BBB) and secretome analysis, we observed that OE-MSCs produce numerous proteins allowing them to cross the endothelial wall. Then, pan-genomic DNA microarrays identified signaling molecules that lesioned mouse hippocampus overexpressed. Among the most upregulated cytokines, both recombinant SPP1/osteopontin and CCL2/MCP-1 stimulate OE-MSC migration whereas only CCL2 exerts a chemotactic effect. Additionally, OE-MSCs express SPP1 receptors but not the CCL2 cognate receptor, suggesting a CCR2-independent pathway through other CCR receptors. These results confirm that OE-MSCs can be attracted by chemotactic cytokines overexpressed in inflamed areas and demonstrate that CCL2 is an important factor that could promote OE-MSC engraftment, suggesting improvement for future clinical trials.

Metabolic changes and inflammation in cultured astrocytes from the 5xFAD mouse model of Alzheimer's disease: Alleviation by pantethine.

Astrocytes play critical roles in central nervous system homeostasis and support of neuronal function. A better knowledge of their response may both help understand the pathophysiology of Alzheimer's disease (AD) and implement new therapeutic strategies. We used the 5xFAD transgenic mouse model of AD (Tg thereafter) to generate astrocyte cultures and investigate the impact of the genotype on metabolic changes and astrocytes activation. Metabolomic analysis showed that Tg astrocytes exhibited changes in the glycolytic pathway and tricarboxylic acid (TCA) cycle, compared to wild type (WT) cells. Tg astrocytes displayed also a prominent basal inflammatory status, with accentuated reactivity and increased expression of the inflammatory cytokine interleukin-1 beta (IL-1ß). Compensatory mechanisms were activated in Tg astrocytes, including: i) the hexose monophosphate shunt with the consequent production of reducing species; ii) the induction of hypoxia inducible factor-1 alpha (HIF-1α), known to protect against amyloid-ß (Aß) toxicity. Such events were associated with the expression by Tg astrocytes of human isoforms of both amyloid precursor protein (APP) and presenilin-1 (PS1). Similar metabolic and inflammatory changes were induced in WT astrocytes by exogenous Aß peptide. Pantethine, the vitamin B5 precursor, known to be neuroprotective and anti-inflammatory, alleviated the pathological pattern in Tg astrocytes as well as WT astrocytes treated with Aß. In conclusion, our data enlighten the dual pathogenic/protective role of astrocytes in AD pathology and the potential protective role of pantethine.

MT5-MMP, just a new APP processing proteinase in Alzheimer's disease?

We have recently identified in a transgenic mouse model of Alzheimer's disease (AD) membrane-type 5-MMP (MT5-MMP) as a new player in Alzheimer's pathogenesis, which displays pro-amyloidogenic features and proteolytic processing of amyloid precursor protein (APP). Another group has reported that MT5-MMP processing of APP may release a novel neurotoxic APP fragment. Although MT5-MMP-mediated APP processing appears to be a key pathogenic step, we hypothesize that MT5-MMP may also contribute to AD pathogenesis through complementary mechanisms that involve the activation of pro-inflammatory pathways and/or APP trafficking.

Vitamin D interacts with Esr1 and Igf1 to regulate molecular pathways relevant to Alzheimer's disease.

BACKGROUND: Increasing evidence suggests a potential therapeutic benefit of vitamin D supplementation against Alzheimer's disease (AD). Although studies have shown improvements in cognitive performance and decreases in markers of the pathology after chronic treatment, the mechanisms by which vitamin D acts on brain cells are multiple and remain to be thoroughly studied. We analyzed the molecular changes observed after 5 months of vitamin D3 supplementation in the brains of transgenic 5xFAD (Tg) mice, a recognized mouse model of AD, and their wild type (Wt) littermates. We first performed a kinematic behavioural examination at 4, 6 and 8 months of age (M4, M6 and M8) followed by a histologic assessment of AD markers. We then performed a comparative transcriptomic analysis of mRNA regulation in the neocortex and hippocampus of 9 months old (M9) female mice. RESULTS: Transcriptomic analysis of the hippocampus and neocortex of both Wt and Tg mice at M9, following 5 months of vitamin D3 treatment, reveals a large panel of dysregulated pathways related to i) immune and inflammatory response, ii) neurotransmitter activity, iii) endothelial and vascular processes and iv) hormonal alterations. The differentially expressed genes are not all direct targets of the vitamin D-VDR pathway and it appears that vitamin D action engages in the crosstalk with estrogen and insulin signaling. The misexpression of the large number of genes observed in this study translates into improved learning and memory performance and a decrease in amyloid plaques and astrogliosis in Tg animals. CONCLUSIONS: This study underlies the multiplicity of action of this potent neurosteroid in an aging and AD-like brain. The classical and non-classical actions of vitamin D3 can act in an additive and possibly synergistic manner to induce neuroprotective activities in a context-specific way.

Cerebrovascular pathology during the progression of experimental Alzheimer's disease.

Clinical and experimental evidence point to a possible role of cerebrovascular dysfunction in Alzheimer's disease (AD). The 5xFAD mouse model of AD expresses human amyloid precursor protein and presenilin genes with mutations found in AD patients. It remains unknown whether amyloid deposition driven by these mutations is associated with cerebrovascular changes. 5xFAD and wild type mice (2 to 12months old; M2 to M12) were used. Thinned skull in vivo 2-photon microscopy was used to determine Aß accumulation on leptomeningeal or superficial cortical vessels over time. Parenchymal microvascular damage was assessed using FITC-microangiography. Collagen-IV and CD31 were used to stain basal lamina and endothelial cells. Methoxy-XO4, Thioflavin-S or 6E10 were used to visualize Aß accumulation in living mice or in fixed brain tissues. Positioning of reactive IBA1 microglia and GFAP astrocytes at the vasculature was rendered using confocal microscopy. Platelet-derived growth factor receptor beta (PDGFRß) staining was used to visualize perivascular pericytes. In vivo 2-photon microscopy revealed Methoxy-XO4(+) amyloid perivascular deposits on leptomeningeal and penetrating cortical vessels in 5xFAD mice, typical of cerebral amyloid angiopathy (CAA). Amyloid deposits were visible in vivo at M3 and aggravated over time. Progressive microvascular damage was concomitant to parenchymal Aß plaque accumulation in 5xFAD mice. Microvascular inflammation in 5xFAD mice presented with sporadic FITC-albumin leakages at M4 becoming more prevalent at M9 and M12. 3D colocalization showed inflammatory IBA1(+) microglia proximal to microvascular FITC-albumin leaks. The number of perivascular PDGFRß(+) pericytes was significantly decreased at M4 in the fronto-parietal cortices, with a trend decrease observed in the other structures. At M9-M12, PDGFRß(+) pericytes displayed hypertrophic perivascular ramifications contiguous to reactive microglia. Cerebral amyloid angiopathy and microvascular inflammation occur in 5xFAD mice concomitantly to parenchymal plaque deposition. The prospect of cerebrovascular pharmacology in AD is discussed.

MT5-MMP Promotes Alzheimer's Pathogenesis in the Frontal Cortex of 5xFAD Mice and APP Trafficking in vitro.

We previously reported that deficiency of membrane-type five matrix metalloproteinase (MT5-MMP) prevents amyloid pathology in the cortex and hippocampus of 5xFAD mice, and ameliorates the functional outcome. We have now investigated whether the integrity of another important area affected in Alzheimer's disease (AD), the frontal cortex, was also preserved upon MT5-MMP deficiency in 4-month old mice at prodromal stages of the pathology. We used the olfactory H-maze (OHM) to show that learning impairment associated with dysfunctions of the frontal cortex in 5xFAD was prevented in bigenic 5xFAD/MT5-MMP-/- mice. The latter exhibited concomitant drastic reductions of amyloid beta peptide (Aß) assemblies (soluble, oligomeric and fibrillary) and its immediate precursor, C99. Simultaneously, astrocyte reactivity and tumor necrosis factor alpha (TNF-α) levels were also lowered. Moreover, MT5-MMP deficiency induced a decrease in N-terminal soluble fragments of amyloid precursor protein (APP), including soluble APPα (sAPPα), sAPPß and the MT5-MMP-linked fragment of 95 kDa, sAPP95. However, the lack of MT5-MMP did not affect the activity of ß- and γ-secretases. In cultured HEKswe cells, transiently expressed MT5-MMP localized to early endosomes and increased the content of APP and Aß40 in these organelles, as well as Aß levels in cell supernatants. This is the first evidence that the pro-amyloidogenic features of MT5-MMP lie, at least in part, on the ability of the proteinase to promote trafficking into one of the amyloidogenic subcellular loci. Together, our data further support the pathogenic role of MT5-MMP in AD and that its inhibition improves the functional and pathological outcomes, in this case in the frontal cortex. These data also support the idea that MT5-MMP could become a novel therapeutic target in AD.