Sphingomyelin-induced inhibition of the plasma membrane calcium ATPase causes neurodegeneration in type A Niemann-Pick disease.

Niemann-Pick disease type A (NPA) is a rare lysosomal storage disorder characterized by severe neurological alterations that leads to death in childhood. Loss-of-function mutations in the acid sphingomyelinase (ASM) gene cause NPA, and result in the accumulation of sphingomyelin (SM) in lysosomes and plasma membrane of neurons. Using ASM knockout (ASMko) mice as a NPA disease model, we investigated how high SM levels contribute to neural pathology in NPA. We found high levels of oxidative stress both in neurons from these mice and a NPA patient. Impaired activity of the plasma membrane calcium ATPase (PMCA) increases intracellular calcium. SM induces PMCA decreased activity, which causes oxidative stress. Incubating ASMko-cultured neurons in the histone deacetylase inhibitor, SAHA, restores PMCA activity and calcium homeostasis and, consequently, reduces the increased levels of oxidative stress. No recovery occurs when PMCA activity is pharmacologically impaired or genetically inhibited in vitro. Oral administration of SAHA prevents oxidative stress and neurodegeneration, and improves behavioral performance in ASMko mice. These results demonstrate a critical role for plasma membrane SM in neuronal calcium regulation. Thus, we identify changes in PMCA-triggered calcium homeostasis as an upstream mediator for NPA pathology. These findings can stimulate new approaches for pharmacological remediation in a disease with no current clinical treatments.

GSK-3ß overexpression causes reversible alterations on postsynaptic densities and dendritic morphology of hippocampal granule neurons in vivo.

Adult hippocampal neurogenesis (AHN) is crucial for the maintenance of hippocampal function. Several neurodegenerative diseases such as Alzheimer's disease (AD) are accompanied by memory deficits that could be related to alterations in AHN. Here, we took advantage of a conditional mouse model to study the involvement of glycogen synthase kinase-3ß (GSK-3ß) overexpression (OE) in AHN. By injecting GFP- and PSD95-GFP-expressing retroviruses, we have determined that hippocampal GSK-3ß-OE causes dramatic alterations in both dendritic tree morphology and post-synaptic densities in newborn neurons. Alterations in previously damaged neurons were reverted by switching off the transgenic system and also by using a physiological approach (environmental enrichment) to increase hippocampal plasticity. Furthermore, comparative morphometric analysis of granule neurons from patients with AD and from GSK-3ß overexpressing mice revealed shared morphological alterations. Taken together, these data indicate that GSK-3ß is crucial for hippocampal function, thereby supporting this kinase as a relevant target for the treatment of AD.

Alzheimer's disease and disseminated mycoses.

Alzheimer's disease (AD) is characterized by the presence in the brain of amyloid plaques and neurofibrillary tangles that provoke neuronal cell death, vascular dysfunction and inflammatory processes. In the present work, we have analyzed the existence of fungal infection in AD patients. A number of tests have been carried out in blood serum, including the detection of antibodies against several yeast species and fungal proteins, and also the presence of fungal (1,3)-ß-glucan. Results from this analysis indicate that there is disseminated fungal infection in the majority of AD patients tested. Of interest, several AD patients contain high levels of fungal polysaccharides in peripheral blood, reflecting that disseminated fungal infection occurs in these patients. Together, these results suggest the presence of disseminated mycoses in blood serum from AD patients. To our knowledge these findings represent the first evidence that fungal infection is detectable in blood samples in AD patients. The possibility that this may represent a risk factor or may contribute to the etiological cause of AD is discussed.

Prolonged fixation and post-mortem delay impede the study of adult neurogenesis in mice.

Adult hippocampal neurogenesis (AHN) gives rise to new neurons throughout life. This phenomenon takes place in more than 120 mammalian species, including humans, yet its occurrence in the latter was questioned after one study proposed the putative absence of neurogenesis markers in the adult human hippocampus. In this regard, we showed that prolonged fixation impedes the visualization of Doublecortin+ immature neurons in this structure, whereas other authors have suggested that a dilated post-mortem delay (PMD) underlies these discrepancies. Nevertheless, the individual and/or additive contribution of fixation and the PMD to the detection (or lack thereof) of other AHN markers has not been studied to date. To address this pivotal question, we used a tightly controlled experimental design in mice, which allowed the dissection of the relative contribution of the aforementioned factors to the visualization of markers of individual AHN stages. Fixation time emerged as the most prominent factor globally impeding the study of this process in mice. Moreover, the visualization of other particularly sensitive epitopes was further prevented by prolonged PMD. These results are crucial to disambiguate current controversies related to the occurrence of AHN not only in humans but also in other mammalian species.

Progression of Alzheimer's disease parallels unusual structural plasticity of human dentate granule cells.

Alzheimer´s disease (AD), the most common form of dementia in industrialized countries, severely targets the hippocampal formation in humans and mouse models of this condition. The adult hippocampus hosts the continuous addition of new dentate granule cells (DGCs) in numerous mammalian species, including humans. Although the morphology and positioning of DGCs within the granule cell layer (GCL) match their developmental origin in rodents, a similar correlation has not been reported in humans to date. Our data reveal that DGCs located in inner portions of the human GCL show shorter and less complex dendrites than those found in outer portions of this layer, which are presumably generated developmentally. Moreover, in AD patients, DGCs show early morphological alterations that are further aggravated as the disease progresses. An aberrantly increased number of DGCs with several primary apical dendrites is the first morphological change detected in patients at Braak-Tau I/II stages. This alteration persists throughout AD progression and leads to generalized dendritic atrophy at late stages of the disease. Our data reveal the distinct vulnerability of several morphological characteristics of DGCs located in the inner and outer portions of the GCL to AD and support the notion that the malfunction of the hippocampus is related to cognitive impairments in patients with AD.

Response to Comment on "Impact of neurodegenerative diseases on human adult hippocampal neurogenesis".

Rakic and colleagues challenge the use of extensively validated adult hippocampal neurogenesis (AHN) markers and postulate an alternative interpretation of some of the data included in our study. In Terreros-Roncal et al., reconstruction of the main stages encompassed by human AHN revealed enhanced vulnerability of this phenomenon to neurodegenerative diseases. Here, we clarify points and ambiguities raised by these authors.

Response to Comment on "Impact of neurodegenerative diseases on human adult hippocampal neurogenesis".

Alvarez-Buylla and colleagues provide an alternative interpretation of some of the data included in our manuscript and question whether well-validated markers of adult hippocampal neurogenesis (AHN) are related to this phenomenon in our study. In Terreros-Roncal et al., reconstruction of the main stages of human AHN revealed its enhanced vulnerability to neurodegeneration. Here, we clarify ambiguities raised by these authors.

Impact of neurodegenerative diseases on human adult hippocampal neurogenesis.

Disrupted hippocampal performance underlies psychiatric comorbidities and cognitive impairments in patients with neurodegenerative disorders. To understand the contribution of adult hippocampal neurogenesis (AHN) to amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, dementia with Lewy bodies, and frontotemporal dementia, we studied postmortem human samples. We found that adult-born dentate granule cells showed abnormal morphological development and changes in the expression of differentiation markers. The ratio of quiescent to proliferating hippocampal neural stem cells shifted, and the homeostasis of the neurogenic niche was altered. Aging and neurodegenerative diseases reduced the phagocytic capacity of microglia, triggered astrogliosis, and altered the microvasculature of the dentate gyrus. Thus, enhanced vulnerability of AHN to neurodegeneration might underlie hippocampal dysfunction during physiological and pathological aging in humans.

Cannabinoid receptor CB2 ablation protects against TAU induced neurodegeneration.

Tauopathies are a group of neurodegenerative diseases characterized by the alteration/aggregation of TAU protein, for which there is still no effective treatment. Therefore, new pharmacological targets are being sought, such as elements of the endocannabinoid system (ECS). We analysed the occurrence of changes in the ECS in tauopathies and their implication in the pathogenesis. By integrating gene expression analysis, immunofluorescence, genetic and adeno-associated virus expressing TAU mouse models, we found a TAU-dependent increase in CB2 receptor expression in hippocampal neurons, that occurs as an early event in the pathology and was maintained until late stages. These changes were accompanied by alterations in the endocannabinoid metabolism. Remarkably, CB2 ablation in mice protects from neurodegeneration induced by hTAUP301L overexpression, corroborated at the level of cognitive behaviour, synaptic plasticity, and aggregates of insoluble TAU. At the level of neuroinflammation, the absence of CB2 did not produce significant changes in concordance with a possible neuronal location rather than its classic glial expression in these models. These findings were corroborated in post-mortem samples of patients with Alzheimer's disease, the most common tauopathy. Our results show that neurons with accumulated TAU induce the expression of the CB2 receptor, which enhances neurodegeneration. These results are important for our understanding of disease mechanisms, providing a novel therapeutic strategy to be investigated in tauopathies.

Human DNA methylomes of neurodegenerative diseases show common epigenomic patterns.

Different neurodegenerative disorders often show similar lesions, such as the presence of amyloid plaques, TAU-neurotangles and synuclein inclusions. The genetically inherited forms are rare, so we wondered whether shared epigenetic aberrations, such as those affecting DNA methylation, might also exist. The studied samples were gray matter samples from the prefrontal cortex of control and neurodegenerative disease-associated cases. We performed the DNA methylation analyses of Alzheimer's disease, dementia with Lewy bodies, Parkinson's disease and Alzheimer-like neurodegenerative profile associated with Down's syndrome samples. The DNA methylation landscapes obtained show that neurodegenerative diseases share similar aberrant CpG methylation shifts targeting a defined gene set. Our findings suggest that neurodegenerative disorders might have similar pathogenetic mechanisms that subsequently evolve into different clinical entities. The identified aberrant DNA methylation changes can be used as biomarkers of the disorders and as potential new targets for the development of new therapies.

Different behavior toward bovine spongiform encephalopathy infection of bovine prion protein transgenic mice with one extra repeat octapeptide insert mutation.

In humans, insert mutations within the repetitive octapeptide region of the prion protein gene (Prnp) are often associated with familial spongiform encephalopathies. In this study, transgenic mice expressing bovine PrP (boTg mice) bearing an additional octapeptide insertion to the wild type (seven octapeptide repeats instead of six) showed an altered course of bovine spongiform encephalopathy (BSE) infection, reflected as reduced incubation times when compared with boTg mice expressing similar levels of the wild-type six-octapeptide protein. In both boTg mouse lines (bo6ORTg and bo7ORTg), incubation times were affected drastically depending on transgene expression levels and the inoculum used. In accordance with the lack of an interspecies barrier to BSE infection, we detected the typical signs of CNS spongiform degeneration by histopathological analysis and the presence of the bovine prion PrP(res) by Western blot or immunohistochemical analyses. When 7OR-PrP(res) was propagated in bo7ORTg mice, a similar earlier onset of clinical signs was observed compared with bo6ORTg mice. Proteins PrP(C) and PrP(res) containing seven octapeptides (7OR-PrP(C) and 7OR-PrP(res)) showed similar protease sensitivity and insolubility in nondenaturing detergents to homologous 6OR-PrP(C) and 6OR-PrP(res). In addition, bo7ORTg mice showed a higher sensitivity than bo6ORTg mice for detecting prion infection in specimens previously diagnosed as negative by conventional biochemical techniques. In the absence of clinical signs of disease, 7OR-PrP(res) could be detected as early as 120 d after inoculation by immunohistochemical and Western blot analyses. These findings may help us improve the current mouse bioassays and understand the role of the octapeptide repeat region in susceptibility to disease.

Transgenic mice expressing bovine PrP with a four extra repeat octapeptide insert mutation show a spontaneous, non-transmissible, neurodegenerative disease and an expedited course of BSE infection.

Transgenic (Tg) mice carrying four extra octapeptide repeats (OR) in the bovine PrP gene (10OR instead of 6) have been generated. In these mice, neuropathological changes were observed depending upon the level of transgene expression. These changes primarily involved a slowly advancing neurological disorder, characterized clinically by ataxia, and neuropathologically, by vacuolization in different brain areas, gliosis, and loss of cerebellar granule cells. Accumulation of insoluble bovine 10OR-PrP (bo10OR-PrP) was observed depending on the level of expression but no infectivity was found associated with this insoluble form. We also compared the behavior of bo6OR-PrP and bo10OR-PrP Tg mouse lines in response to BSE infection. BSE-inoculated bo10ORTg mice showed an altered course of BSE infection, reflected by reduced incubation times when compared to bo6ORTg mice expressing similar levels of the wild type 6OR-PrP. In BSE-inoculated mice, it was possible to detect PrP(res) in 100% of the animals. While insoluble bo10OR-PrP from non-inoculated bo10ORTg mice was non-infectious, brain homogenates from BSE-inoculated bo10ORTg mice were highly infectious in all the Tg mouse lines tested. This Tg mouse model constitutes a new way of understanding the pathobiology of bovine transmissible spongiform encephalopathy. Its potential applications include the assessment of new therapies against prion diseases.

Fatty acid amide hydrolase localization in the human central nervous system: an immunohistochemical study.

Recent discoveries have opened new fields for research on the biochemistry and pharmacology of cannabinoids. Among them, and most importantly, are the characterization and molecular cloning of central and peripheral cannabinoid receptors as well as the isolation of the first putative endogenous ligands that bind to them, anandamide and 2-arachidonylglycerol. The enzyme that degrades these so-called "endocannabinoids" is an integral membrane protein, fatty acid amide hydrolase. Its distribution and biochemistry in rat brain suggest that it plays a critical role in the regulation of the endocannabinoid system. However, few data exist regarding its distribution and mechanism of action in human tissues. To that end, we have studied its cellular distribution in the human central nervous system by immunohistochemistry. Using an affinity-purified antibody, we report that fatty acid amide hydrolase is localized to specific and well-delimited cell populations, including cortical pyramidal neurons, subcortical white matter astrocytes, striatal and striatoefferent projecting neurons, hypothalamic and midbrain nuclei, granular and molecular layers of the cerebellum, Purkinje neurons, dentate cerebellar nucleus, inferior olivary nuclei and others. This distribution resembles that of the central cannabinoid receptors as well as that of the enzyme distribution in the rat brain. In summary, the cellular localization of the degradative enzyme of the endogenous cannabinoid ligands in human central nervous system reveals its presence on both neuronal and glial elements and shows a significant overlapping with that of central cannabinoid receptors, mainly in areas related with motor control, confirming the notion that the endocannabinoid system plays a critical role in the control of movement.

Effects of cicaprost and fosinopril on the progression of rat diabetic nephropathy.

We have studied the effects of chronic therapy with cicaprost (a PGI2 analog), fosinopril (a converting enzyme inhibitor), and the combination of both drugs on the progression of experimental diabetic nephropathy. Uninephrectomized streptozotocin-induced diabetic rats were maintained for 8 months with plasma glucose between 13.7 and 22.0 mmol/L to hasten renal damage. Systemic and renal parameters were measured periodically, and at sacrifice structural and morphometrical renal studies were performed to evaluate diabetic injury. Control rats exhibited characteristic features of this model, such as high blood pressure and plasma creatinine and urinary albumin excretion, together with prominent alterations in the kidney (renal and glomerular hypertrophies, mesangial matrix expansion, and tubular alterations). The three therapies attenuated equivalently the progression of diabetic renal injury, as estimated by lower urinary albumin excretion, renal and glomerular hypertrophies, and a better renal architectural preservation. No synergistic action was appreciated with the combined therapy. However, renal preservation achieved with cicaprost was not linked to reductions in systemic blood pressure, whereas in the groups treated with fosinopril the hypotensive effect of this drug could have contributed to the positive outcome of the therapy. Therefore, nephroprotection exerted by this PGI2 analog in this model seems more related to the derangement of renal local mechanisms than to systemic blood pressure control. Finally, the possibility that an impaired prostacyclin synthesis or bioavailability is involved in the pathogenesis of the diabetic nephropathy in this model underlies our results.

Effects of chronic combined treatment with captopril and pravastatin on the progression of insulin resistance and cardiovascular alterations in an experimental model of obesity in dogs.

Obesity is a metabolic disorder in which multiple clinical and biochemical alterations coexist. However, the progression of these alterations in relation to weight gain has not been investigated in detail. Therefore, we studied the evolution of insulin resistance and associated risk factors in a model of experimental obesity in dogs. We also studied whether chronic exposure to these pathogenic factors could induce cardiac and vascular alterations. Twenty male age- and body weight-matched beagle dogs were divided into four groups (n = 5), according to diet and pharmacologic therapy received, and followed for 2 years. Control animals were maintained with a regular diet, while the 15 remaining animals were fed a high-fat diet. The Obese group of dogs received no therapy, whereas the Capto group received 25 mg/12 h captopril, and the Prava+Capto was treated with 10 mg/24 h pravastatin plus the same dose of captopril throughout the study. Periodical determinations of clinical and biochemical parameters were made, and the degree of insulin resistance was also estimated. After the 2-year follow-up, the dogs were killed and vascular thickening in the aorta and the coronary arteries was evaluated. In addition, cardiac hypertrophy was estimated by heart weight and free-wall left ventricular width. Chronic pravastatin plus captopril treatment, together with decreasing weight gain rate, ameliorated the progression of insulin resistance and associated risk factors (hyperinsulinemia, hypercholesterolemia) related to this severe model. In addition, this combined therapy showed cardioprotective action, as cardiac and vascular hypertrophy observed in the Obese group was prevented. These positive results seems to emerge from the synergistic effects of both drugs, as captopril as monotherapy induced only a slight benefit on these parameters.

Effects of UP269-6, a new angiotensin II receptor antagonist, and captopril on the progression of rat diabetic nephropathy.

To estimate the effects of UP269-6, a nonpeptide angiotensin II receptor antagonist, and captopril, a converting enzyme inhibitor, on the progression of nephropathy, 77 uninephrectomized diabetic rats were maintained for 8 months with plasma glucose levels from 300 to 500 mg/dL. Systemic and renal parameters were periodically measured, and, at the time of death, a histological evaluation of renal damage was performed. Control rats (no additional treatment but insulin) showed increased blood pressure and urinary albumin levels, together with prominent alterations in the kidney (renal and glomerular hypertrophies, tubular atrophy, and 19% of sclerotic glomeruli). Captopril (50 mg/kg/day) and UP269-6 (10 mg/kg/day) reduced blood pressure and albumin excretion levels, and improved histological renal preservation (lower renal and glomerular hypertrophies, tubular atrophy, and percentage of sclerotic glomeruli: 5% and 7%, respectively). Finally, a low dose of UP269-6 (1 mg/kg/day), which induced an intermediate level of blood pressure between control and the other treated groups, produced an equivalent degree of nephroprotection. Our data demonstrate the efficacy of this new angiotensin II receptor antagonist on the progression of diabetic renal damage. These results also reinforce the role attributed to angiotensin II in the development of renal derangement in this model, as UP269-6 is devoid of agonistic effect on the kinin system.

Effects of hyperinsulinemia on vascular blood flows in experimental obesity.

Human obesity, which is very common in Polycystic Ovaries Syndrome and in "X Syndrome", constitutes an insulin-resistance state in which multiple clinical, biochemical and hemodynamic alterations coexist. Insulin resistance in the obese has been recently associated with an endothelial dysfunction. To investigate the possibility that clinical and metabolic derangements related to insulin resistance could induce changes in vascular blood flows, we have studied the levels of mesenteric (MBF), renal (RBF) and femoral (FBF) blood flows in Beagle dogs kept for 2 years on a normal (control group) or high fat diet (obese group). This experimental model exhibits many of the abnormalities with the human syndrome. In addition, we have tested the effects of chronic treatment with captopril (capto group) in monotherapy or in association with pravastatin (prava+capto group) on the hemodynamic changes associated with this diet. After the two year follow-up, Transonic flow probes were placed around the three arteries to measure basal blood flows and their response to a hyperinsulinemic-normoglycemic test in anesthetized animals. During this test the degree of insulin sensitivity was estimated. In association with higher body weight, blood pressure, insulin resistance, and fasting levels of insulin and total cholesterol, the obese group exhibited decreased basal levels of FBF and a greater femoral vasoconstriction during hyperinsulinism (P < 0.05 vs control). Combined therapy with captopril and pravastatin ameliorated the reduction in basal FBF and hyperinsulinism-induced vasoconstriction (P < 0.05), in addition to the beneficial effects on insulin sensitivity, and clinical and metabolic parameters. Synergistic beneficial effects of both drugs on lipid and carbohydrate profiles may account for this positive outcome, by attenuating the atherogenic process associated with this model.

Long-term intracerebral infusion of fibroblast growth factors restores motility and enhances F-DOPA uptake in parkinsonian monkeys.

Fibroblast growth factors (FGFs) are important for dopamine neurons in health and disease. Acidic (aFGF) and basic (bFGF) fibroblast growth factors increase the survival and growth of dopamine cells. Nigrostriatal dopamine neurons, the target cells for degeneration in Parkinson's disease, display receptors for basic fibroblast growth factor and these receptors are decreased in the brain of parkinsonian patients. We have investigated the effects of long-term intrastriatal infusion of FGFs in hemiparkinsonian monkeys. All animals were lesioned with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 0.4mgkg(-1), into the left internal carotid artery. The monkeys that had persistent asymmetric akinesia and contralateral rotation induced by apomorphine, were selected for chronic, unilateral, intracerebral infusion of neurotrophic factors or vehicle into the striatum ipsilateral to the lesion. Two animals received intrastriatal aFGF or bFGF, 2mugweek(-1), for 6 months. The controls received intrastriatal saline or intraventricular epidermal growth factor (EGF). F-DOPA positron emission tomography scans were performed in each animal before and after the intracerebral infusion of neurotrophic factors. We measured the tyrosine hydroxylase (TH) immunoreactive neurons in the substantia nigra and terminals in the striatum and evaluated the pathological complications related to the treatment or the delivery system. All four animals had, after the lesion with MPTP, a transient but incomplete recovery of akinesia. This period of spontaneous improvement was followed by a progressive deterioration of motor behaviour during the following months. The monkeys treated with FGFs, however, recovered quickly and persistently during the intracerebral infusion. F-DOPA uptake, prior to the intracerebral infusion, was greatly reduced in the lesioned striatum. The post-infusion F-DOPA scans revealed a 60% reduction respect to baseline in the lesioned striatum of the saline and EGF-infused animals. In the animals infused with FGFs, the post-infusion F-DOPA uptake increased more than 400% in the lesioned (and infused) striatum and around 200-300% in the contralateral side, with respect to the pre-infusion scan. The number of TH-positive cells in the substantia nigra correlated well with the uptake of F-DOPA in the post-infusion scan. No severe side-effects were present. Intrastriatal infusion of FGFs restores motor behaviour and increases F-DOPA striatal uptake in hemiparkinsonian monkeys.

Emboli in bulls killed in Spanish traditional bullfighting.

The finding of brain tissue fragments in blood and lungs of cattle stunned in slaughterhouses has raised concerns about food safety in the context of the bovine spongiform encephalopathy epidemic. In the present study, the possible occurrence of brain tissue emboli in animals killed in traditional Spanish bullfighting was investigated. Thorough histological analysis of multiple possible target organs was carried out in 434 bulls. No evidence of brain tissue embolism was obtained, but emboli from diverse sources were detected in pulmonary and hepatic tissue of a significant number of animals. These emboli seem to have been caused by the use of a long sword, which extensively disrupts intra-thoracic and intra-abdominal organs and vascular structures.