Olga Touloumi

Activity-Dependent Neuroprotective Protein Snippet NAP Reduces Tau Hyperphosphorylation and Enhances Learning in a Novel Transgenic Mouse Model

Activity-dependent neuroprotective protein (ADNP) differentially interacts with chromatin to regulate essential genes. Because complete ADNP deficiency is embryonic lethal, the outcome of partial ADNP deficiency was examined. ADNP+/– mice exhibited cognitive deficits, significant increases in phosphorylated tau, tangle-like structures, and neurodegeneration compared with ADNP+/+ mice. Increased tau hyperphosphorylation is known to cause memory impairments in neurodegenerative diseases associated with tauopathies, including the most prevalent Alzheimers disease. The current results suggest that ADNP is an essential protein for brain function and plays a role in normal cognitive performance. ADNP-deficient mice offer an ideal paradigm for evaluation of cognitive enhancers. NAP (NAPVSIPQ) is a peptide derived from ADNP that interacts with microtubules and provides potent neuroprotection. NAP treatment partially ameliorated cognitive deficits and reduced tau hyperphosphorylation in the ADNP+/– mice. NAP is currently in phase II clinical trials assessing effects on mild cognitive impairment.

Variable behavior and complications of autologous bone marrow mesenchymal stem cells transplanted in experimental autoimmune encephalomyelitis

Autologous bone marrow stromal cells (BMSCs) offer significant practical advantages for potential clinical applications in multiple sclerosis (MS). Based on recent experimental data, a number of clinical trials have been designed for the intravenous (IV) and/or intrathecal (ITH) administration of BMSCs in MS patients. Delivery of BMSCs in the cerebrospinal fluid via intracerebroventricular (ICV) transplantation is a useful tool to identify mechanisms underlying the migration and function of these cells. In the current study, BMSCs were ICV administered in severe and mild EAE, as well as naive animals; neural precursor cells (NPCs) served as cellular controls. Our data indicated that ICV-transplanted BMSCs significantly ameliorated mild though not severe EAE. Moreover, BMSCs exerted significant anti-inflammatory effect on spinal cord with concomitant reduced axonopathy only in the mild EAE model. BMSCs migrated into the brain parenchyma and, depending on their cellular density, within brain parenchyma formed cellular masses characterized by focal inflammation, demyelination, axonal loss and increased collagen-fibronectin deposition. These masses were present in 64% of ICV BMASC-transplanted severe EAE animals whereas neither BMSCs transplanted in mild EAE cases nor the NPCs exhibited similar behavior. BMSCs possibly exerted their fibrogenic effect via both paracrine and autocrine manner, at least partly due to up-regulation of connective tissue growth factor (CTGF) under the trigger of TGFb1. Our findings are of substantial relevance for clinical trials in MS, particularly regarding the possibility that ICV transplanted BMSCs entering the inflamed central nervous system may exhibit - under conditions - a local pathology of yet unknown consequences.

Statins reduce the neurofibrillary tangle burden in a mouse model of tauopathy.

Abstract Statin treatment has been associated with a reduced risk of Alzheimer disease and decreased amyloid deposition in mouse models. No animal studies have reported effects of statins on tau aggregates and neurofibrillary tangles (NFTs), the pathological hallmarks of Alzheimer disease that correlate with dementia. We investigated the effect of statins on NFTs in a transgenic mouse tauopathy model and found the following: 1) 1-month treatment with the blood-brain barrier-permeable agent simvastatin in normocholesterolemic aged mice significantly reduced the NFT burden and decreased lectin-positive microglia; 2) simvastatin significantly decreased NFTs and improved T-maze performance in young animals treated for 8 months; 3) treatment of hypercholesterolemic mice for 5 months with blood-brain barrier-impermeable atorvastatin markedly reduced the NFT burden and decreased lectin-positive microglia; 4) nonstatin cholesterol-lowering strategies showed a modest NFT decrease compared with statin treatment; and 5) there was a positive correlation between microglial and NFT burden (r = 0.8). Together, these results suggest that statins reduce NFT burden irrespective of blood-brain barrier permeability at both early and late ages in long- and short-term treatment paradigms and under normocholesterolemic and hypercholesterolemic conditions. The decrease in microglia, coupled with the limited effect of nonstatin cholesterol lowering, suggests that the anti-NFT effect of statins may be related to their anti-inflammatory and not necessarily to their cholesterol-lowering properties. Statins may provide therapy against NFTs in tauopathies, particularly when NFTs are the major neuropathologic component.

Administration of 2-arachidonoylglycerol ameliorates both acute and chronic experimental autoimmune encephalomyelitis.

BACKGROUND AND PURPOSE Experimental autoimmune encephalomyelitis (EAE) is a widely used model of multiple sclerosis (MS) and both conditions have been reported to exhibit reduced endocannabinoid activity. The purpose of this study was to address the effect of exogenously administered 2-arachidonoylglycerol (2AG), an endocannabinoid receptor ligand, on acute phase and chronic disability in EAE. EXPERIMENTAL APPROACH Acute and chronic EAE models were induced in susceptible mice and 2AG-treatment was applied for 14 days from day of disease induction. KEY RESULTS 2AG-treatment ameliorated acute phase of disease with delay of disease onset in both EAE models and reduced disease mortality and long-term (70 days post-induction) clinical disability in chronic EAE. Reduced axonal pathology in the chronic EAE- (p<0.0001) and increased activation and ramification of microglia in the 2AG-treated acute EAE- (p<0.05) model were noticed. The latter was accompanied by a 2- to 4-fold increase of the M2-macrophages in the perivascular infiltrations (p<0.001) of the 2AG-treated animals in the acute (day 22), although not the chronic (day 70), EAE model. Expression of cannabinoid receptors 1 (CB1R) and 2 (CB2R) was increased in 2AG-treated animals of acute EAE vs. controls (p<0.05). In addition, ex vivo viability assays exhibited reduced proliferation of activated lymph node cells when extracted from 2AG-treated EAE animals, whereas a dose-dependent response of activated lymphocytes to 2AG-treatment in vitro was noticed. CONCLUSION AND IMPLICATIONS Our data indicate for the first time that 2AG treatment may provide direct (via CBRs) and immune (via M2 macrophages) mediated neuroprotection in EAE.

Davunetide (NAP) as a preventative treatment for central nervous system complications in a diabetes rat model

AIMS Central nervous system complications including cognitive impairment are an early manifestation of diabetes mellitus, also evident in animal models. NAP (generic name, davunetide), a neuroprotective peptide was tested here for its ability to prevent diabetes-related brain pathologies in the streptozotocin injected diabetes rat model. METHODS Diabetes was induced by an intraperitoneal streptozotocin injection (55 mg/kg). Intranasal NAP or vehicle was administered daily starting on the day following streptozotocin injection. Cognitive assessment was performed 12 weeks after diabetes induction, using the Morris water maze paradigm. Brain structural integrity was assessed on the 15th week of diabetes by magnetic resonance T2 scan. Characterization of cellular populations, apoptosis and synaptic density was performed 16 weeks after diabetes induction, using immunohistochemical markers and quantified in the prefrontal cortex, the cerebral cortex and the hippocampus of both hemispheres. RESULTS Impaired spatial memory of the diabetic rats was observed in the water maze by attenuated learning curve and worsened performance in the probe memory test. NAP treatment significantly improved both measurements. T2 magnetic resonance imaging revealed atrophy in the prefrontal cortex of the diabetes rat group, which was prevented by NAP treatment. Immunohistochemical analysis showed that NAP treatment protected against major loss of the synaptic marker synaptophysin and astrocytic apoptosis, resulting from streptozotocin treatment. CONCLUSIONS Our results show for the first time protective effects for NAP (davuentide) in a diabetes rat model at the behavioral and structural levels against one of the most severe complications of diabetes.

Autophagy and neurodegenerative disorders

Accumulation of aberrant proteins and inclusion bodies are hallmarks in most neurodegenerative diseases. Consequently, these aggregates within neurons lead to toxic effects, overproduction of reactive oxygen species and oxidative stress. Autophagy is a significant intracellular mechanism that removes damaged organelles and misfolded proteins in order to maintain cell homeostasis. Excessive or insufficient autophagic activity in neurons leads to altered homeostasis and influences their survival rate, causing neurodegeneration. The review article provides an update of the role of autophagic process in representative chronic and acute neurodegenerative disorders.

Time Course and Spatial Profile of Nogo-A Expression in Experimental Autoimmune Encephalomyelitis in C57BL/6 Mice

Abstract Inhibition of the myelin-associated neurite outgrowth inhibitor Nogo-A has been found to be beneficial in experimental autoimmune encephalomyelitis (EAE), but there are little data on its expression dynamics during the disease course. We analyzed Nogo-A mRNA and protein during the course of EAE in 27 C57BL/6 mice and in 8 controls. Histopathologic and molecular analyses were performed on Day 0 (naive), preclinical (Day 10), acute (Days 18–22) and chronic (Day 50) time points. In situ hybridization and real-time polymerase chain reaction analyses revealed reduced Nogo-A mRNA expression at preclinical (p < 0.0001) and acute phases (p < 0.0001), followed by upregulation during the chronic phase (p < 0.0001). Nogo-A mRNA was expressed in neurons and oligodendrocytes. By immunohistochemistry and Western blot, there was increased Nogo-A protein expression (p < 0.001) in the chronic phase. Moreover, spatial differences were observed within EAE lesions. The pattern of Nogo-A protein expression inversely correlated with axonal regeneration growth-associated protein 43–positive axons (60% of which were Nogo-A contact–free during the acute phase) and axonal injury (&bgr;-amyloid precursor protein–positive axons). Cortical Nogo-66 receptor protein and mRNA levels increased during the chronic phase. The results indicate that Nogo-A and Nogo receptor are actively regulated in EAE lesions; this may indicate a specific time window for localized axonal regeneration in the acute phase of EAE.

Microglial involvement in neuroprotection following experimental traumatic brain injury in heat-acclimated mice.

Brain-derived neurotrophic factor (BDNF) conveys neuroprotection in various settings of experimental central nervous system injury. Using a model of endogenous neuroprotection, induced in mice by chronic exposure to moderate ambient heat (heat acclimation, HA), we have previously shown that neuroprotection following traumatic brain injury involves reduced post-injury tumor necrosis factor alpha (TNFalpha) expression. As glial cells play a pivotal role in post-injury inflammation on one hand, and are also capable of inducing neuroprotection by harboring trophic factors and BDNF in particular, the effects of injury and HA on overall BDNF content at the trauma area, gliosis and glial BDNF expression were investigated. Western blotting indicated higher overall BDNF levels in HA sham-operated mice. Following injury, a decrease was observed in the HA group only, reaching levels similar to normothermic mice. Immunohistochemical studies demonstrated BDNF-positive resting microglia in non-injured HA but not normothermic animals. Post-injury astrocytosis and microglial immunoreactivity were enhanced in the HA group. Particularly, an increase in the amount of ramified microglia was observed within the penumbra, accompanied by a concomitant decrease in globular microglia, a major source of pro-inflammatory mediators. BDNF labeling on and around microglia and their processes was intensified in HA mice. Furthermore, BDNF immunoreactivity in HA mice was evident in the degenerated edges of axons. These findings, taken together with the growing body of evidence indicating the neuroprotective potential of both BDNF and microglia, suggest a possible role of these cells in HA-induced neuroprotection.

New horizons in schizophrenia treatment: autophagy protection is coupled with behavioral improvements in a mouse model of schizophrenia

Autophagy plays a key role in the pathophysiology of schizophrenia as manifested by a 40% decrease in BECN1/Beclin 1 mRNA in postmortem hippocampal tissues relative to controls. This decrease was coupled with the deregulation of the essential ADNP (activity-dependent neuroprotector homeobox), a binding partner of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β) another major constituent of autophagy. The drug candidate NAP (davunetide), a peptide fragment from ADNP, enhanced the ADNP-LC3B interaction. Parallel genetic studies have linked allelic variation in the gene encoding MAP6/STOP (microtubule-associated protein 6) to schizophrenia, along with altered MAP6/STOP protein expression in the schizophrenic brain and schizophrenic-like behaviors in Map6-deficient mice. In this study, for the first time, we reveal significant decreases in hippocampal Becn1 mRNA and reversal by NAP but not by the antipsychotic clozapine (CLZ) in Map6-deficient (Map6+/−) mice. Normalization of Becn1 expression by NAP was coupled with behavioral protection against hyperlocomotion and cognitive deficits measured in the object recognition test. CLZ reduced hyperlocomotion below control levels and did not significantly affect object recognition. The combination of CLZ and NAP resulted in normalized outcome behaviors. Phase II clinical studies have shown NAP-dependent augmentation of functional activities of daily living coupled with brain protection. The current studies provide a new mechanistic pathway and a novel avenue for drug development.

Immunomodulation of EAE by alpha-fetoprotein involves elevation of immune cell apoptosis markers and the transcription factor FoxP3.

Alpha-fetoprotein (AFP) is an immunomodulatory glycoprotein associated with the normal growth of the mammalian fetus. Ws have shown that treatment with recombinant human AFP (rhAFP) reduced lymphocyte reactivity and the extent of neuroinflammation in mice with experimental autoimmune encephalomyelitis (EAE). In the present study we found involvement of AFP in immune cell apoptosis, attesting to its possible mechanism of action. AFP increased the expression of the Bax, Bid, Bad and ApaF genes in peripheral lymphocytes, together with an enhanced expression of Caspase-3, Fas, FasL and TRAIL among infiltrating immune cells. The induction of apoptosis markers was accompanied with an increased expression of Foxp3 in lymph node cells, as well as accumulation of CD4+Foxp3+ regulatory T cells in the CNS. Overall, these immunological alterations gave rise to a milder disease and accelerated remission rate. Our results suggest a new role for AFP in controlling the autoimmune inflammation associated with EAE.