Amyloid Precursor Protein and Tau Peptides Linked Together Ameliorate Loss of Cognition in an Alzheimer's Disease Animal Model.

The major proteins involved in Alzheimer's disease (AD) are amyloid precursor protein (APP) and Tau. We demonstrate that APP1 (390-412) and Tau1 (19-34), linked together with either a flexible or a rigid peptide bridge, are able to inhibit, in vitro, the interaction between APP and Tau proteins. Furthermore, nasal administration of biotin-labelled Flex peptide for two weeks indicated the localization of the peptide around and close to plaques in the hippocampus area. In vivo studies in 5xFAD transgenic (Tg) mice, which exhibit plaque load and mild cognitive decline at four months of age, show that nasal administration of the flexible linked peptide reduced amyloid plaque burden. Additionally, nasal treatment with either flexible or rigid linked peptides prevented cognitive function deterioration. A significant treatment effect was achieved when either treatment was initiated at the age of three months, before severe cognitive deficiency is evident, or at five months, when such deficiency is already observed. The nasally treated mice demonstrated a cognitive ability not significantly different from the non-Tg littermate controls. Testing the effect of the flexible peptide by gavage feeding on the cognitive function of 5xFAD Tg mice demonstrated that feeding as well as nasal treatment significantly improves the cognitive ability of Tg mice compared to control PBS-treated mice.

Role of Helical Structure in MBP Immunodominant Peptides for Efficient IgM Antibody Recognition in Multiple Sclerosis.

The involvement of Myelin Basic Protein (MBP) in Multiple Sclerosis (MS) has been widely discussed in the literature. This intrinsically disordered protein has an interesting α-helix motif, which can be considered as a conformational epitope. In this work we investigate the importance of the helical structure in antibody recognition by MBP peptides of different lengths. Firstly, we synthesized the peptide MBP (81-106) (1) and observed that its elongation at both N- and C-termini, to obtain the peptide MBP (76-116) (2) improves IgM antibody recognition in SP-ELISA, but destabilizes the helical structure. Conversely, in competitive ELISA, MBP (81-106) (1) is recognized more efficiently by IgM antibodies than MBP (76-116) (2), possibly thanks to its more stable helical structure observed in CD and NMR conformational experiments. These results are discussed in terms of different performances of peptide antigens in the two ELISA formats tested.

Neuroprotective Effect of Glatiramer Acetate on Neurofilament Light Chain Leakage and Glutamate Excess in an Animal Model of Multiple Sclerosis.

Axonal and neuronal pathologies are a central constituent of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), induced by the myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide. In this study, we investigated neurodegenerative manifestations in chronic MOG 35-55 induced EAE and the effect of glatiramer acetate (GA) treatment on these manifestations. We report that the neuronal loss seen in this model is not attributed to apoptotic neuronal cell death. In EAE-affected mice, axonal damage prevails from the early disease phase, as revealed by analysis of neurofilament light (NFL) leakage into the sera along the disease duration, as well as by immunohistological examination. Elevation of interstitial glutamate concentrations measured in the cerebrospinal fluid (CSF) implies that glutamate excess plays a role in the damage processes inflicted by this disease. GA applied as a therapeutic regimen to mice with apparent clinical symptoms significantly reduces the pathological manifestations, namely apoptotic cell death, NFL leakage, histological tissue damage, and glutamate excess, thus corroborating the neuroprotective consequences of this treatment.

Astrocytes in Multiple Sclerosis-Essential Constituents with Diverse Multifaceted Functions.

In multiple sclerosis (MS), astrocytes respond to the inflammatory stimulation with an early robust process of morphological, transcriptional, biochemical, and functional remodeling. Recent studies utilizing novel technologies in samples from MS patients, and in an animal model of MS, experimental autoimmune encephalomyelitis (EAE), exposed the detrimental and the beneficial, in part contradictory, functions of this heterogeneous cell population. In this review, we summarize the various roles of astrocytes in recruiting immune cells to lesion sites, engendering the inflammatory loop, and inflicting tissue damage. The roles of astrocytes in suppressing excessive inflammation and promoting neuroprotection and repair processes is also discussed. The pivotal roles played by astrocytes make them an attractive therapeutic target. Improved understanding of astrocyte function and diversity, and the mechanisms by which they are regulated may lead to the development of novel approaches to selectively block astrocytic detrimental responses and/or enhance their protective properties.

Titration of myelin oligodendrocyte glycoprotein (MOG) - Induced experimental autoimmune encephalomyelitis (EAE) model.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) induced by the myelin oligodendrocyte glycoprotein (MOG) peptide 35-55, is a widely used multiple sclerosis (MS) model. Unlike the spontaneous occurrence of MS, in EAE, external immunization with the MOG peptide (200-300 µg/mouse), emulsified in adjuvant enriched with Mycobacterium Tuberculosis (MT) H37Ra (100-500 µg mouse), and pertussis toxin (PTx, 200-500 ng/mouse) injections, are applied, which heavily boosts the immune system. NEW METHOD: A detailed and systematic titration of the MOG 35-55 EAE induction protocol in C57BL/6 mice reveals the minimal doses of the MOG 35-55 peptide, MT H37Ra, and PTx, required for disease manifestation. RESULTS: The amounts of MOG 35-55 peptide, MT H37Ra, and PTx can be drastically reduced from the standard protocol, to level of 5 µg MOG, 25 µg MT H37Ra, and 50 ng PTx, without affecting the clinical manifestations. The titrated protocols induced a high disease incidence and a consistent robust disease course, with full histopathological characteristics of the MOG model, inflammation, demyelination and axonal damage. COMPARISON WITH EXISTING METHODS: Similar disease incidences, day of symptoms appearance, maximal clinical score, and histopathology were obtained for the standard and the titrated protocols. CONCLUSIONS: Reducing the reagent dosages used for EAE induction, without attenuating the disease, can give a truer and less artificial perspective of MS. We propose an improved protocol for this extensively used model, with high disease incidence, a consistent robust course, and characteristic histological manifestations, which may be more sensitive for testing therapeutic modalities, cost-effective, and less distressing to the animals.

Glatiramer acetate increases T- and B -regulatory cells and decreases granulocyte-macrophage colony-stimulating factor (GM-CSF) in an animal model of multiple sclerosis.

To identify the mechanisms relevant for the therapeutic effect of glatiramer acetate (GA), we studied T- and B- regulatory cells as well as GM-CSF expression in mice recovered from experimental autoimmune encephalomyelitis (EAE). Selective depletion of Tregs reduced but did not eliminate the ability of GA to ameliorate EAE, indicating a role for additional immune-subsets. The prevalence of Bregs in the periphery and the CNS of EAE-mice increased following GA-treatment. Furthermore, GA downregulated the pathological expression of GM-CSF, on both the protein and mRNA levels. These findings corroborate the broad immunomodulatory mechanism of action of GA in EAE/MS.

Peptide Interference with APP and Tau Association: Relevance to Alzheimer's Disease Amelioration.

The two major proteins involved in Alzheimer's disease (AD) are the amyloid precursor protein (APP) and Tau. Here, we demonstrate that these two proteins can bind to each other. Four possible peptides APP1 (390-412), APP2 (713-730), Tau1 (19-34) and Tau2 (331-348), were predicted to be involved in this interaction, with actual binding confirmed for APP1 and Tau1. In vivo studies were performed in an Alzheimer Disease animal model-APP double transgenic (Tg) 5xFAD-as well as in 5xFAD crossed with Tau transgenic 5xFADXTau (FT), which exhibit declined cognitive reduction at four months of age. Nasal administration of APP1 and Tau1 mixture, three times a week for four or five months, reduced amyloid plaque burden as well as the level of soluble Aß 1-42 in the brain. The treatment prevented the deterioration of cognitive functions when initiated at the age of three months, before cognitive deficiency was evident, and also at the age of six months, when such deficiencies are already observed, leading to a full regain of cognitive function.

Cognitive impairment in an animal model of multiple sclerosis and its amelioration by glatiramer acetate.

The severe motor impairment in the MS animal model experimental autoimmune encephalomyelitis (EAE) obstructs the assessment of cognitive functions. We developed an experimental system that evaluates memory faculties in EAE-affected mice, irrespective of their motor performance, enabling the assessment of cognitive impairments along the disease duration, the associated brain damage, and the consequences of glatiramer acetate (GA) treatment on these manifestations. The delayed-non-matching to sample (DNMS) T-maze task, testing working and long term memory was adapted and utilized. Following the appearance of clinical manifestations task performances of the EAE-untreated mice drastically declined. Cognitive impairments were associated with disease severity, as indicated by a significant correlation between the T-maze performance and the clinical symptoms in EAE-untreated mice. GA-treatment conserved cognitive functions, so that despite their exhibited mild motor impairments, the treated mice performed similarly to naïve controls. The cognitive deficit of EAE-mice coincided with inflammatory and neurodegenerative damage to the frontal cortex and the hippocampus; these damages were alleviated by GA-treatment. These combined findings indicate that in addition to motor impairment, EAE leads to substantial impairment of cognitive functions, starting at the early stages and increasing with disease aggravation. GA-treatment, conserves cognitive capacities and prevents its disease related deterioration.

Glatiramer Acetate: from Bench to Bed and Back.

BACKGROUND: Glatiramer acetate (GA, Copaxone®, Copolymer1, Cop 1) is an approved drug for the treatment of relapsing-remitting multiple sclerosis (RRMS). Its efficacy in reducing the frequency of exacerbations and its safety profile establish it as a first-line therapy for MS. Evidence from the animal model experimental autoimmune encephalomyelitis (EAE) and from MS patients indicate that GA affects various levels of the innate and the adaptive immune response, inducing deviation from the pro-inflammatory to the anti-inflammatory pathways. This includes mainly the induction of Th2/3 and T-regulatory cells, and down-regulation of both Th1 and Th17 cells. The immune cells induced by GA reach the CNS and secrete in situ anti-inflammatory cytokines, alleviating the pathological processes. In addition to its immunomodulatory activities, GA promotes neuroprotective repair processes such as secretion of neurotrophic factors, remyelination and neurogenesis, indicating that repair process in the CNS can be up-regulated by therapy.

Astrocyte disruption of neurovascular communication is linked to cortical damage in an animal model of multiple sclerosis.

To elucidate mechanisms contributing to cortical pathology in multiple sclerosis (MS), we investigated neurovascular aberrations, in particular the association of astrocytes with cortical neurons and blood vessels, in mice induced with experimental autoimmune encephalomyelitis (EAE). Blood-brain barrier (BBB) dysfunction was evident by leakage of the tracer sodium fluorescein, along with reduced expression of claudin-5 by endothelial cells and desmin by pericytes. Immunohistological and ultrastructural analyses revealed detachment of the astroglial cell bodies from the blood vessels and loss of their connections with both the blood vessels and the neuronal synapses. Furthermore, examination of individual astrocytic processes at cortical layer IV, where well-defined neuronal columns (barrels) are linked to functional properties, revealed loss of astrocytic confinement to the functional neuronal boundaries. Thus, in contrast to the highly modulated patches of astrocyte processes in naïve mice overlapping the barrel cores, in EAE-mice process distribution was uniform ignoring the barrel boundaries. These aberrations are attributed to the surrounding inflammation, indicated by T-cells presence in the cortex as well as in the subcortical white matter and the meninges. Immunomodulatory treatment with glatiramer acetate partially abrogated the neurovascular damage. These combined findings indicate that under inflammatory conditions, activated perivascular astrocytes fail in neuro-hemodynamic coupling, resulting in obstructed cross-talk between the blood vessels and the neurons. We propose that loss of cortical astrocytic regulation and fine-tuning between the blood supply and the neuronal needs contributes to the neurological impairment and cognitive decline occurring in EAE/MS as well as to the disease progression.

Assessing remyelination - metabolic labeling of myelin in an animal model of multiple sclerosis.

The lack of biomarkers is a major obstacle for investigating myelin repair. We used metabolic incorporation of the choline analog - propargyl-choline (P-Cho) to label and visualize newly synthesized myelin in the CNS of mice induced with experimental autoimmune encephalomyelitis (EAE). We further developed unbiased colocalization analysis to quantify P-Cho incorporation specifically into the myelin. Our findings indicate that P-Cho injection to mice recovering from EAE, either spontaneously or following glatiramer acetate treatment, results in significant elevation of its incorporation into the myelin, offering a novel strategy for assessing remyelination in animal models and the remyelination potential of candidate drugs.

Structural Transition in Myelin Membrane as Initiator of Multiple Sclerosis.

In demyelinating diseases such as multiple sclerosis, disrupted myelin structures impair the functional role of the sheath as an insulating layer for proper nerve conduction. Though the etiology and recovery pathways remain unclear, in vivo studies show alterations in the lipid and the adhesive protein (myelin basic protein, MBP) composition. We find that in vitro cytoplasmic myelin membranes with modified lipid composition and low MBP concentration, as in demyelinating disease, show structural instabilities and pathological phase transition from a lamellar to inverted hexagonal, which involve enhanced local curvature. Similar curvatures are also found in vivo in diseased myelin sheaths. In addition, MBP dimers form a correlated mesh-like network within the inner membrane space, only in the vicinity of native lipid composition. These findings delineate the distinct functional roles of dominant constituents in cytoplasmic myelin sheaths, and shed new light on mechanisms disrupting lipid-protein complexes in the diseased state.

Astrocyte morphology is confined by cortical functional boundaries in mammals ranging from mice to human.

Cortical blood flow can be modulated by local activity across a range of species; from barrel-specific blood flow in the rodent somatosensory cortex to the human cortex, where BOLD-fMRI reveals numerous functional borders. However, it appears that the distribution of blood capillaries largely ignores these functional boundaries. Here we report that, by contrast, astrocytes, a major player in blood-flow control, show a striking morphological sensitivity to functional borders. Specifically, we show that astrocyte processes are structurally confined by barrel boundaries in the mouse, by the border of primary auditory cortex in the rat and by layers IIIa/b and Cytochrome Oxidase (CO)-blobs boundaries in the human primary visual cortex. Thus, astrocytes which are critical elements in neuro-hemodynamic coupling show a significant anatomical segregation along functional boundaries across different mammalian species. These results may open a new anatomical marker for delineating functional borders across species, including post-mortem human brains.

Perforin-Positive Dendritic Cells Exhibit an Immuno-regulatory Role in Metabolic Syndrome and Autoimmunity.

Emerging evidence suggests that immunological mechanisms underlie metabolic control of adipose tissue. Here, we have shown the regulatory impact of a rare subpopulation of dendritic cells, rich in perforin-containing granules (perf-DCs). Using bone marrow transplantation to generate animals selectively lacking perf-DCs, we found that these chimeras progressively gained weight and exhibited features of metabolic syndrome. This phenotype was associated with an altered repertoire of T cells residing in adipose tissue and could be completely prevented by T cell depletion in vivo. A similar impact of perf-DCs on inflammatory T cells was also found in a well-defined model of multiple sclerosis, experimental autoimmune encephlalomyelitis (EAE). Thus, perf-DCs probably represent a regulatory cell subpopulation critical for protection from metabolic syndrome and autoimmunity.

Sequential capsule endoscopy of the small bowel for follow-up of patients with known Crohn's disease.

BACKGROUND AND AIMS: The aim of this study was to perform sequential small bowel (SB) capsule endoscopy (CE) studies in patients with known active Crohn's disease (CD) during different treatments, to characterize the changes in the SB mucosa over time, and to correlate the CE findings with clinical and laboratory parameters of inflammation. METHODS: Consecutive patients with known moderately active CD were prospectively recruited. After proven patency with Agile capsule, CE studies were performed at baseline and after 4, 12 and 24 weeks. CE parameters and a Lewis score were calculated. Clinical and laboratory parameters were correlated. A control group of 178 non-CD patients was used for comparisons. RESULTS: Thirty-one CD patients were recruited and 19 met the inclusion criteria. A total of 43 CE studies were performed over the time. There was no capsule retention despite a high rate of previous SB surgery. The mean baseline CDAI, IBDQ and Lewis scores were 306±56, 135±26.6 and 1730±1780, respectively. There was no correlation at the baseline between clinical and laboratory parameters (CDAI, CRP, IBDQ) and mucosal disease (Lewis scores). CDAI and IBDQ changes over a period of 4 and 12 weeks did not correlate with the Lewis score. The cecum arrival rate of the CD patients was significantly lower (p=0.0047) and the SB transit time was significantly longer (p=0.005) compared to those of the controls. CONCLUSIONS: Sequential CE studies are feasible and safe in CD patients. In patients with complete CE studies, they provide reliable information on mucosal changes in CD and should be considered as an independent and objective follow-up tool in known CD patients.

Oligodendrogenesis and myelinogenesis during postnatal development effect of glatiramer acetate.

Myelinogenesis in the mammal nervous system occurs predominantly postnatally. Glatiramer acetate (GA), a drug for the treatment for multiple sclerosis (MS), has been shown to induce immunomodulation and neuroprotection in the inflamed CNS in MS and in experimental autoimmune encephalomyelitis (EAE). Here we investigated whether GA can affect myelinogenesis and oligodendrogenesis in the developing nervous system under nonpathological conditions. Towards this end we studied myelination in mice injected daily by GA, at postnatal Days 7-21. Immunohistological and ultrastructural analyses revealed significant elevation in the number of myelinated axons as well as in the thickness of the myelin encircling them and their resulting g-ratios, in spinal cords of GA-injected mice compared with their PBS-injected littermates, at postnatal Day 14. Elevation in myelinated axons was detected also in the peripheral ventral roots of the motor nerves. GA induced also an increase in axonal diameter, implying an effect on the overall development of the nervous system. A prominent elevation in the amount of progenitor oligodendrocytes and their BrdU incorporation, as well as in mature oligodendrocytes indicated that the effect of GA is linked to increased proliferation and differentiation along the oligodendroglial maturation cascade. In addition, elevation in insulin-like growth factor (IGF-1) and brain-derived neurotrophic factor (BDNF) was found in the white matter of the GA-injected mice. Furthermore, a functional advantage in rotating rod test was exhibited by GA-injected mice over their littermates at postnatal Day 21. These cumulative findings corroborate the beneficial effect of GA on oligodendrogenesis and myelination.

Age dependent course of EAE in Aire-/- mice.

This study explores the consequences of deficiency in the autoimmune regulator (Aire) on the susceptibility to experimental autoimmune encephalomyelitis (EAE). Increased susceptibility to EAE was found in Aire knockout (KO) compared to wild type (WT) in 6month old mice. In contrast, 2month old Aire KO mice were less susceptible to EAE than WT mice, and this age-related resistance correlated with elevated proportions of T regulatory (Treg) cells in their spleen and brain. Combined with our previous findings in experimental autoimmune myasthenia gravis, we suggest an age-related association between Aire and Treg cells in the susceptibility to autoimmunity.

Magnetic resonance imaging characterization of different experimental autoimmune encephalomyelitis models and the therapeutic effect of glatiramer acetate.

The roles of inflammation and degeneration as well as of gray matter abnormalities in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are controversial. We analyzed the pathological manifestations in two EAE models, the chronic oligodendrocyte glycoprotein (MOG)-induced versus the relapsing-remitting proteolipid protein (PLP)-induced, along the disease progression, using advanced magnetic resonance imaging (MRI) parameters. The emphasis of this study was the overall assessment of the whole brain by histogram analysis, as well as the detection of specific affected regions by voxel based analysis (VBA) using quantitative T2, magnetization transfer ratio (MTR) and diffusion tensor imaging (DTI). Brains of EAE-inflicted mice from both models revealed multiple white and gray matter areas with significant changes from naïve mice for all MRI parameters. Ventricle swelling was more characteristic to the PLP-induced model. Decreased MTR values and increased apparent diffusion coefficient (ADC) were observed mainly in MOG-induced EAE, indicative of macromolecular loss and structural CNS damage involvement in the chronic disease. The MS drug glatiramer acetate (GA), applied either as prevention or therapeutic treatment, affected all the MRI pathological manifestations, resulting in reduced T2 values and ventricle volume, elevated MTR and decreased ADC, in comparison to untreated EAE-inflicted mice. In accord, immunohistochemical analysis indicated less histological damage and higher amount of proliferating oligodendrocyte progenitor cells after GA treatment. The higher brain tissue integrity reflected by the MRI parameters on the level of the whole brain and in specific regions supports the in situ anti-inflammatory and neuroprotective consequences of GA treatment.