Isabel Carreras

Sodium phenylbutyrate prolongs survival and regulates expression of anti-apoptotic genes in transgenic amyotrophic lateral sclerosis mice

Multiple molecular defects trigger cell death in amyotrophic lateral sclerosis (ALS). Among these, altered transcriptional activity may perturb many cellular functions, leading to a cascade of secondary pathological effects. We showed that pharmacological treatment, using the histone deacetylase inhibitor sodium phenylbutyrate, significantly extended survival and improved both the clinical and neuropathological phenotypes in G93A transgenic ALS mice. Phenylbutyrate administration ameliorated histone hypoacetylation observed in G93A mice and induced expression of nuclear factor‐κB (NF‐κB) p50, the phosphorylated inhibitory subunit of NF‐κB (pIκB) and beta cell lymphoma 2 (bcl‐2), but reduced cytochrome c and caspase expression. Curcumin, an NF‐κB inhibitor, and mutation of the NF‐κB responsive element in the bcl‐2 promoter, blocked butyrate‐induced bcl‐2 promoter activity. We provide evidence that the pharmacological induction of NF‐κB‐dependent transcription and bcl‐2 gene expression is neuroprotective in ALS mice by inhibiting programmed cell death. Phenylbutyrate acts to phosphorylate IκB, translocating NF‐κB p50 to the nucleus, or to directly acetylate NF‐κB p50. NF‐κB p50 transactivates bcl‐2 gene expression. Up‐regulated bcl‐2 blocks cytochrome c release and subsequent caspase activation, slowing motor neuron death. These transcriptional and post‐translational pathways ultimately promote motor neuron survival and ameliorate disease progression in ALS mice. Phenylbutyrate may therefore provide a novel therapeutic approach for the treatment of patients with ALS.

Ibuprofen reduces Aβ, hyperphosphorylated tau and memory deficits in Alzheimer mice

We examined the effects of ibuprofen on cognitive deficits, Abeta and tau accumulation in young triple transgenic (3xTg-AD) mice. 3xTg-AD mice were fed ibuprofen-supplemented chow between 1 and 6 months. Untreated 3xTg-AD mice showed significant impairment in the ability to learn the Morris water maze (MWM) task compared to age-matched wild-type (WT) mice. The performance of 3xTg-AD mice was significantly improved with ibuprofen treatment compared to untreated 3xTg-AD mice. Ibuprofen-treated transgenic mice showed a significant decrease in intraneuronal oligomeric Abeta and hyperphosphorylated tau (AT8) immunoreactivity in the hippocampus. Confocal microscopy demonstrated co-localization of conformationally altered (MC1) and early phosphorylated tau (CP-13) with oligomeric Abeta, and less co-localization of oligomeric Abeta and later forms of phosphorylated tau (AT8 and PHF-1) in untreated 3xTg-AD mice. Our findings show that prophylactic treatment of young 3xTg-AD mice with ibuprofen reduces intraneuronal oligomeric Abeta, reduces cognitive deficits, and prevents hyperphosphorylated tau immunoreactivity. These findings provide further support for intraneuronal Abeta as a cause of cognitive impairment, and suggest that pathological alterations of tau are associated with intraneuronal oligomeric Abeta accumulation.

Channel formation by serum amyloid A: a potential mechanism for amyloid pathogenesis and host defense

Serum amyloid A (SAA) is a family of closely related apolipoproteins associated with high density lipoprotein (HDL). Subclasses of SAA isoforms are differentially expressed constitutively and during inflammation. During states of infection or inflammation, levels of HDL, bound, acute phase isoforms of SAA rise as much as 1000-fold in the serum, suggesting that it might play a role in host defense. Following recurrent or chronic inflammation, an N-terminalpeptide fragment of SAA known as amyloid A (AA) assembles into fibrils causing extensive damage to spleen, liver, and kidney, and rapidly progressing to death. In the present paper, we report the novel finding that a recombi-nant acute phase isoform variant of human SAA1.1 (SAAp) readily forms ion-channels in planar liptd bilayer membranes at physiologic concentrations. These channels are voltage-independent, poorly selective, and are relatively long-lived. This type of channel would place a severe metabolic strain on various kinds of cells. Expression of human SAA 1.1 in bacteria induces lysis of bacterial cells, while expression of the constitutive isoform (human SAA4) does not. Secondary structural analysis of the SAA isoforms indicates a strong hydrophobicity of the N-terminal of the acute phase isoform relative to the constitutive SAA4 isoform, which may be responsible for the bactericidal activity of the former, in keeping with the notion that SAA 1 targets cell membranes and forms channels in them. Channel formation may thus be related to a host defense role of acute phase SAA isoforms and may also be the mechanism of end organ damage in AA and other amyloidoses.

Anti-inflammatory treatment in AD mice protects against neuronal pathology

Prior studies suggest that non-steroidal anti-inflammatory drugs (NSAIDs) may lower the incidence of Alzheimers disease (AD) and delay onset or slow progression of symptoms in mouse models of AD. We examined the effects of chronic NSAID treatment in order to determine which elements of the pathological features might be ameliorated. We compared the effects of the NSAIDs ibuprofen and celecoxib on immunohistological and neurochemical markers at two different ages in APPxPS1 mice using measurements of amyloid plaque deposition, Abeta peptide levels, and neurochemical profiles using magnetic resonance spectroscopy (MRS). At 6 months of age, few neurochemical changes were observed between PSAPP mice and WT mice using MRS. Ibuprofen, but not celecoxib, treatment significantly decreased the Abeta(42/40) ratio in frontal cortex at 6 months, but overall amyloid plaque burden was unchanged. Consistent with prior findings in mouse models, at 17 months of age, there was a decrease in the neuronal markers NAA and glutamate and an increase in the astrocytic markers glutamine and myo-inositol in AD mice compared to WT. Ibuprofen provided significant protection against NAA and glutamate loss. Neither of the drugs significantly affected myo-inositol or glutamine levels. Both ibuprofen and celecoxib lowered plaque burden without a significant effect on Abeta(1-42) levels. NAA levels significantly correlated with plaque burden. These results suggest that selective NSAIDs (ibuprofen and possibly celecoxib) treatment can protect against the neuronal pathology.

Moderate exercise delays the motor performance decline in a transgenic model of ALS

The relationship between exercise and amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by motor neuron loss, rapidly progressive weakness and early death has been controversial. We studied the effect of a high (HEX) and moderate-level exercise (MEX) on body weight, motor performance and motor neuron counts in the ventral horn of spinal cords in a transgenic mouse model of ALS (G93A-SOD1) that overexpresses a mutated form of the human SOD1 gene that is a cause of familial ALS. These transgenic mice show several similarities to the human disease, including rapid progressive motor weakness from 100 days of age and premature death at around 135 days of age. Mice were exposed to high or mid-level exercise of left sedentary (SED). At 70, 95 and 120 days of age, spinal cords were processed following euthanasia. Motor neurons larger than 15 mum in diameter were counted with a design-based stereological protocol using an optical fractionator probe in the ventral horn of different regions of the cord and compared to wild-type littermates. Moderate exercise delayed the onset of motor deficit by over a week. High exercise slightly but significantly hastened the onset of motor performance deficits. Motor neuron density in the lumbar cord was significantly higher in MEX group compared to SED at 95 days of age. These results show the beneficial effects of moderate exercise on the preservation of motor performance that correlates with higher motor neuron density in the ventral horn of the lumbar spinal cord in G93A mice.

Insulin degrading enzyme is localized predominantly at the cell surface of polarized and unpolarized human cerebrovascular endothelial cell cultures

Insulin degrading enzyme (IDE) is expressed in the brain and may play an important role there in the degradation of the amyloid beta peptide (Aβ). Our results show that cultured human cerebrovascular endothelial cells (HCECs), a primary component of the blood–brain barrier, express IDE and may respond to exposure to low levels of Aβ by upregulating its expression. When radiolabeled Aβ is introduced to the medium of cultured HCECs, it is rapidly degraded to smaller fragments. We believe that this degradation is largely the result of the action of IDE, as it can be substantially blocked by the presence of insulin in the medium, a competitive substrate of IDE. No inhibition is seen when an inhibitor of neprilysin, another protease that may degrade Aβ, is present in the medium. Our evidence suggests that the action of IDE occurs outside the cell, as inhibitors of internalization fail to affect the rate of the observed degradation. Further, our evidence suggests that degradation by IDE occurs on the plasma membrane, as much of the IDE present in HCECs was biotin‐labeled by a plasma membrane impermeable reagent. This activity seems to be polarity dependent, as measurement of Aβ degradation by each surface of differentiated HCECs shows greater degradation on the basolateral (brain‐facing) surface. Thus, IDE could be an important therapeutic target to decrease the amount of Aβ in the cerebrovasculature.

Intraperitoneal injection of the pancreatic peptide amylin potently reduces behavioral impairment and brain amyloid pathology in murine models of Alzheimer's disease.

Amylin, a pancreatic peptide, and amyloid-beta peptides (Aβ), a major component of Alzheimer’s disease (AD) brain, share similar β-sheet secondary structures, but it is not known whether pancreatic amylin affects amyloid pathogenesis in the AD brain. Using AD mouse models, we investigated the effects of amylin and its clinical analog, pramlintide, on AD pathogenesis. Surprisingly, chronic intraperitoneal (i.p.) injection of AD animals with either amylin or pramlintide reduces the amyloid burden as well as lowers the concentrations of Aβ in the brain. These treatments significantly improve their learning and memory assessed by two behavioral tests, Y maze and Morris water maze. Both amylin and pramlintide treatments increase the concentrations of Aβ1-42 in cerebral spinal fluid (CSF). A single i.p. injection of either peptide also induces a surge of Aβ in the serum, the magnitude of which is proportionate to the amount of Aβ in brain tissue. One intracerebroventricular injection of amylin induces a more significant surge in serum Aβ than one i.p. injection of the peptide. In 330 human plasma samples, a positive association between amylin and Aβ1-42 as well as Aβ1-40 is found only in patients with AD or amnestic mild cognitive impairment. As amylin readily crosses the blood–brain barrier, our study demonstrates that peripheral amylin’s action on the central nervous system results in translocation of Aβ from the brain into the CSF and blood that could be an explanation for a positive relationship between amylin and Aβ in blood. As naturally occurring amylin may play a role in regulating Aβ in brain, amylin class peptides may provide a new avenue for both treatment and diagnosis of AD.

Basic fibroblast growth factor decreases elastin gene transcription in aortic smooth muscle cells

The extracellular matrix (ECM) protein elastin plays an essential role in the cardiovascular system by imparting elasticity to blood vessel wall. In this study, we examined the effect of basic fibroblast growth factor (bFGF) on the expression of elastin in aortic smooth muscle cells (SMC) to gain insight into events associated with cardiovascular diseases. The results show that bFGF treatment of SMC causes a significant decrease in elastin mRNA and secreted tropoelastin levels. Nuclear run‐on analyses demonstrate that the downregulation is due to a decrease in the level of elastin gene transcription. Transient transfections of SMC with wild‐type and mutated elastin gene promoter/chloramphenicol acetyl transferase (CAT) constructs show that a previously identified activator protein‐1‐cAMP response element (AP1/CRE) (−564 to −558‐bp) within the elastin promoter mediates the bFGF‐dependent downregulation of elastin gene transcription in SMC. Addition of bFGF to SMC activates the extracellular signal‐regulated kinases 1/2 (ERK1/2) resulting in their translocation into the nucleus and subsequent induction of Fra‐1. The addition of PD‐98059, an inhibitor of ERK1/2 kinase, abrogates the bFGF‐dependent decrease of elastin mRNA in SMC. The described inhibitory effect of bFGF on elastin gene expression in SMC may significantly contribute to the inefficient repair of elastin in early stages of vascular wall injury. J. Cell. Biochem. 85: 592–600, 2002.

Combination therapy in a transgenic model of Alzheimer’s disease

The pathological accumulation of the β-amyloid protein (Aβ) has been closely associated with synaptic loss and neurotoxicity contributing to cognitive dysfunction in Alzheimers disease (AD). Oligomers of Aβ42 appear to be the most neurotoxic form. Two of the most promising attempts to reduce Aβ accumulation have been with scyllo-inositol, an inositol steroisomer, that stabilizes Aβ42 peptide and prevents it from progressing to oligomers and fibrils and R-flurbiprofen, a purified enantiomer of the classical racemic non-steroidal anti-inflammatory drugs (NSAID), flurbiprofen, that retains the ability to specifically lower Aβ42. In the present study we evaluated the effects of scyllo-inositol and the combination treatment of scyllo-inositol+R-flurbiprofen on amyloid pathology and hippocampal-dependent memory function in 5XFAD mice, a model of Aβ pathology characterized by an enormous production of Aβ42. Our expectations were that the combination treatment of scyllo-inositol+R-flurbiprofen would have an additive effect in preventing Aβ accumulation and that cognition would be improved. Mice treated with scyllo-inositol exhibit 41 and 35% reduction in the deposition of the amyloid plaques stained by antibody against Aβ42 and Aβ40 respectively. Scyllo-inositol was not more effective when combined with R-flurbiprofen for the measures tested. Scyllo-inositol treated mice performed significantly better at the radial arm water maze (RAWM) task than untreated and scyllo-inositol+R-flurbiprofen treated mice.

7,8-Dihydroxyflavone improves motor performance and enhances lower motor neuronal survival in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an enigmatic neurodegenerative disorder without any effective treatment characterized by loss of motor neurons (MNs) that results in rapidly progressive motor weakness and early death due to respiratory failure. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family known to play a prominent role in the differentiation and survival of MNs. The flavonoid 7,8-dihydroxyflavone (7,8-DHF) is a potent and selective small molecule tyrosine kinase receptor B (TrkB) agonist that mimics the effects of BDNF. In the present study, we evaluated the neuroprotective effects of 7,8-DHF in a transgenic ALS mouse model (SOD1(G93A)). We found that chronic administration of 7,8-DHF significantly improved motor deficits, and preserved spinal MNs count and dendritic spines in SOD1(G93A) mice. These data suggest that 7,8-DHF should be considered as a potential therapy for ALS and the other motor neuron diseases.