Ischemia induces cell proliferation and neurogenesis in the gerbil hippocampus in response to neuronal death

We studied hippocampal cellular proliferation and neurogenesis processes in a model of transient global cerebral ischemia in gerbils by labelling dividing cells with 5'-Bromo-2'-deoxyuridine (BrdU). Surrounding the region of selective neuronal death (CA1 pyramidal layer of the hippocampus), an important increase in reactive astrocytes and BrdU-labelled cells was detected 5 days after ischemia. A similar result was found in the dentate gyrus (DG) 12 days after ischemia. The differentiation of the BrdU+ cells was investigated 28 days after BrdU administration by analyzing the morphology, anatomic localization and cell phenotype by triple fluorescent labelling (BrdU, adult neural marker NeuN and DNA marker TOPRO-3) using confocal laser-scanning microscopy. This analysis showed increased neurogenesis in the DG in case of ischemia and triple positive labelling in some newborn cells in CA1. Seven brain hemispheres from gerbils subjected to ischemia did not develop CA1 neuronal death; hippocampus from these hemispheres did not show any of the above mentioned findings. Our results indicate that ischemia triggers proliferation in CA1 and neurogenesis in the DG in response to CA1 pyramidal neuronal death, independently of the reduced cerebral blood flow or the cell migration from subventricular zone (SVZ)

Tramiprosate, a drug of potential interest for the treatment of Alzheimer's disease, promotes an abnormal aggregation of tau

Alzheimer's disease (AD) is characterized by the presence of two histopathological hallmarks; the senile plaques, or extracellular deposits mainly composed of amyloid-β peptide (Aβ), and the neurofibrillary tangles, or intraneuronal inclusions composed of hyperphosphorylated tau protein

Dysregulated protein phosphorylation: A determining condition in the continuum of brain aging and Alzheimer's disease

Tau hyperphosphorylation is the first step of neurofibrillary tangle (NFT) formation. In the present study, samples of the entorhinal cortex (EC) and frontal cortex area 8 (FC) of cases with NFT pathology classified as stages I-II, III-IV, and V-VI without comorbidities, and of middle-aged (MA) individuals with no NFT pathology, were analyzed by conventional label-free and SWATH-MS (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) to assess the (phospho)proteomes. The total number of identified dysregulated phosphoproteins was 214 in the EC, 65 of which were dysregulated at the first stages (I-II) of NFT pathology; 167 phosphoproteins were dysregulated in the FC, 81 of them at stages I-II of NFT pathology. A large percentage of dysregulated phosphoproteins were identified in the two regions and at different stages of NFT progression. The main group of dysregulated phosphoproteins was made up of components of the membranes, cytoskeleton, synapses, proteins linked to membrane transport and ion channels, and kinases. The present results show abnormal phosphorylation of proteins at the first stages of NFT pathology in the elderly (in individuals clinically considered representative of normal aging) and sporadic Alzheimer's disease (sAD). Dysregulated protein phosphorylation in the FC precedes the formation of NFTs and SPs. The most active period of dysregulated phosphorylation is at stages III-IV when a subpopulation of individuals might be clinically categorized as suffering from mild cognitive impairment which is a preceding determinant stage in the progression to dementia. Altered phosphorylation of selected proteins, carried out by activation of several kinases, may alter membrane and cytoskeletal functions, among them synaptic transmission and membrane/cytoskeleton signaling. Besides their implications in sAD, the present observations suggest a molecular substrate for "benign" cognitive deterioration in "normal" brain aging

Enhanced expression of the voltage-dependent anion channel 1 (VDAC1) in Alzheimer's disease transgenic mice: an insight into the pathogenic effects of amyloid-β

The mitochondrial voltage-dependent anion channel 1 (VDAC1) is involved in the release of apoptotic proteins with possible relevance in Alzheimer's disease (AD) neuropathology. Through proteomic analysis followed by Western blotting and immunohistochemical techniques, we have found that VDAC1 is overexpressed in the hippocampus from amyloidogenic AD transgenic mice models. VDAC1 was also overexpressed in postmortem brain tissue from AD patients at an advanced stage of the disease. Interestingly, amyloid-β (Aβ) soluble oligomers were able to induce upregulation of VDAC1 in a human neuroblastoma cell line, further supporting a correlation between Aβ levels and VDAC1 expression. In hippocampal extracts from transgenic mice, a significant increase was observed in the levels of VDAC1 phosphorylated at an epitope that is susceptible to phosphorylation by glycogen synthase kinase-3β, whose activity was also increased. The levels of hexokinase I (HXKI), which interacts with VDAC1 and affects its function, were decreased in mitochondrial samples from AD models. Since phospho-VDAC and reduced HXKI levels favors a VDAC1 conformational state more prone to the release proapoptotic factors, regulation of the function of this channel may be a promising therapeutic approach to combat AD

Enhanced anxiety, depressive-like behaviour and impaired recognition memory in mice with reduced expression of the vesicular glutamate transporter 1 (VGLUT1)

Three isoforms of a vesicular glutamate transporter (VGLUT1-3) have been identified. Of these, VGLUT1 is the major isoform of the cerebral cortex and hippocampus where it is selectively located on synaptic vesicles of excitatory glutamatergic terminals. Variations in VGLUT1 expression levels have a major impact on the efficacy of glutamate synaptic transmission. Given evidence linking alterations in glutamate neurotransmission to various neuropsychiatric disorders, we investigated the possible influence of a down-regulation of VGLUT1 transporter on anxiety, depressive-like behaviour and learning. The behavioural phenotype of VGLUT1 heterozygous mice (C57BL/6) was compared to WT littermates. Moreover, VGLUT1-3 expression, hippocampal excitatory terminal ultrastructure and neurochemical phenotype were analysed. VGLUT1 heterozygous mice displayed normal spontaneous locomotor activity, increased anxiety in the light-dark exploration test and depressive-like behaviour in the forced swimming test: no differences were shown in the elevated plus-maze model of anxiety. In the novel object recognition test, VGLUT1+/- mice showed normal short-term but impaired long-term memory. Spatial memory in the Morris water maze was unaffected. Western blot analysis confirmed that VGLUT1 heterozygotes expressed half the amount of transporter compared to WT. In addition, a reduction of the reserve pool of synaptic vesicles of hippocampal excitatory terminals and a 35-45 % reduction of GABA in the frontal cortex and the hippocampus were observed in the mutant mice. These observations suggest that a VGLUT1-mediated presynaptic alteration of the glutamatergic synapses, in specific brain regions, leads to a behavioural phenotype resembling certain aspects of psychiatric and cognitive disorders

Dysregulated Brain Protein Phosphorylation Linked to Increased Human Tau Expression in the hTau Transgenic Mouse Model

Altered protein phosphorylation is a major pathologic modification in tauopathies and Alzheimer's disease (AD) linked to abnormal tau fibrillar deposits in neurofibrillary tangles (NFTs) and pre-tangles and beta-amyloid deposits in AD. hTau transgenic mice, which express 3R and less 4R human tau with no mutations in a murine knock-out background, show increased tau deposition in neurons but not NFTs and pre-tangles at the age of nine months. Label-free (phospho)proteomics and SWATH-MS identified 2065 proteins in hTau and wild-type (WT) mice. Only six proteins showed increased levels in hTau; no proteins were down-regulated. Increased tau phosphorylation in hTau was detected at Ser199, Ser202, Ser214, Ser396, Ser400, Thr403, Ser404, Ser413, Ser416, Ser422, Ser491, and Ser494, in addition to Thr181, Thr231, Ser396/Ser404, but not at Ser202/Thr205. In addition, 4578 phosphopeptides (corresponding to 1622 phosphoproteins) were identified in hTau and WT mice; 64 proteins were differentially phosphorylated in hTau. Sixty proteins were grouped into components of membranes, membrane signaling, synapses, vesicles, cytoskeleton, DNA/RNA/protein metabolism, ubiquitin/proteasome system, cholesterol and lipid metabolism, and cell signaling. These results showed that over-expression of human tau without pre-tangle and NFT formation preferentially triggers an imbalance in the phosphorylation profile of specific proteins involved in the cytoskeletal-membrane-signaling axis

Proteostatic modulation in brain aging without associated Alzheimer's disease-and age-related neuropathological changes

(Phospho)proteomics of old-aged subjects without cognitive or behavioral symptoms, and without AD-neuropathological changes and lacking any other neurodegenerative alteration will increase understanding about the physiological state of human brain aging without associate neurological deficits and neuropathological lesions.(Phospho)proteomics using conventional label-free- and SWATH-MS (Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) has been assessed in the frontal cortex (FC) of individuals without NFTs, senile plaques (SPs) and age-related co-morbidities classified by age (years) in four groups; group 1 (young, 30-44); group 2 (middle-aged: MA, 45-52); group 3 (early-elderly, 64-70); and group 4 (late-elderly, 75-85).Protein levels and deregulated protein phosphorylation linked to similar biological terms/functions, but involving different individual proteins, are found in FC with age. The modified expression occurs in cytoskeleton proteins, membranes, synapses, vesicles, myelin, membrane transport and ion channels, DNA and RNA metabolism, ubiquitin-proteasome-system (UPS), kinases and phosphatases, fatty acid metabolism, and mitochondria. Dysregulated phosphoproteins are associated with the cytoskeleton, including microfilaments, actin-binding proteins, intermediate filaments of neurons and glial cells, and microtubules; membrane proteins, synapses, and dense core vesicles; kinases and phosphatases; proteins linked to DNA and RNA; members of the UPS; GTPase regulation; inflammation; and lipid metabolism. Noteworthy, protein levels of large clusters of hierarchically-related protein expression levels are stable until 70. However, protein levels of components of cell membranes, vesicles and synapses, RNA modulation, and cellular structures (including tau and tubulin filaments) are markedly altered from the age of 75. Similarly, marked modifications occur in the larger phosphoprotein clusters involving cytoskeleton and neuronal structures, membrane stabilization, and kinase regulation in the late elderly.Present findings may increase understanding of human brain proteostasis modifications in the elderly in the subpopulation of individuals not having AD neuropathological change and any other neurodegenerative change in any telencephalon region

Chronic stress and impaired glutamate function elicit a depressive-like phenotype and common changes in gene expression in the mouse frontal cortex

Major depression might originate from both environmental and genetic risk factors. The environmental chronic mild stress (CMS) model mimics some environmental factors contributing to human depression and induces anhedonia and helplessness. Mice heterozygous for the synaptic vesicle protein (SVP) vesicular glutamate transporter 1 (VGLUT1) have been proposed as a genetic model of deficient glutamate function linked to depressive-like behaviour. Here, we aimed to identify, in these two experimental models, gene expression changes in the frontal cortex, common to stress and impaired glutamate function. Both VGLUT1+/- and CMS mice showed helpless and anhedonic-like behavior. Microarray studies in VGLUT1+/- mice revealed regulation of genes involved in apoptosis, neurogenesis, synaptic transmission, protein metabolic process or learning and memory. In addition, RT-PCR studies confirmed gene expression changes in several glutamate, GABA, dopamine and serotonin neurotransmitter receptors. On the other hand, CMS affected the regulation of 147 transcripts, some of them involved in response to stress and oxidoreductase activity. Interestingly, 52 genes were similarly regulated in both models. Specifically, a dowregulation in genes that promote cell proliferation (Anapc7), cell growth (CsnK1g1), cell survival (Hdac3), inhibition of apoptosis (Dido1) was observed. Genes linked to cytoskeleton (Hspg2, Invs), psychiatric disorders (Grin1, MapK12) or an antioxidant enzyme (Gpx2) were also downregulated. Moreover, genes that inhibit the MAPK pathways (Dusp14), stimulate oxidative metabolism (Eif4a2) and enhance glutamate transmission (Rab8b) were upregulated. We suggest that these genes could form part of the altered “molecular context” underlying depressive-like behaviour in animal models. The clinical relevance of these findings is discussed

Increased vulnerability to depressive-like behaviour of mice with decreased expression of VGLUT1

Background: Many studies have linked depression to an increase in the excitatory-inhibitory ratio in the forebrain. Presynaptic alterations in a shared pathway of the glutamate/GABA cycle may account for this imbalance. Recent evidence suggests that decreased vesicular glutamate transporter 1 (VGLUT1) levels in the forebrain affects the glutamate/GABA cycle and induces helpless behaviour. Here we studied decreased VGLUT1 as a potencial factor enhancing a depressive-like phenotype in an animal model. Methods: Glutamate and GABA synthesis as well as oxidative metabolism were studied in heterozygous mice for the vesicular glutamate transporter 1 (VGLUT1+/-) and WT. Subsequently, the regulation of neurotransmitter levels, proteins involved in the glutamate/GABA cycle and behaviour by both genotype and chronic mild stress (CMS) was studied. Finally, the effect of chronic imipramine on VGLUT1 control and CMS mice was also studied. Results: VGLUT1+/- mice showed increased neuronal synthesis of glutamate, decreased cortical and hippocampal GABA, VGLUT1 and EAAT1, as well as helplessness and anhedonia. CMS induced an increase of glutamate and a decrease of GABA, VGAT and GAD65 in both areas and led to upregulation EAAT1 in the hippocampus. Moreover, CMS induced anhedonia, helplessness, anxiety and impaired recognition memory. VGLUT1+/- CMS mice showed a combined phenotype (genotype plus stress) and specific alterations, such as an upregulation of VGLUT2 and hyperlocomotion. Moreover, an increased vulnerability to anhedonia and helplessness reversible by chronic imipramine was shown. Conclusions: These studies highlight a crucial role for decreased VGLUT1 in the forebrain as a biological mediator of increased vulnerability to chronic mild stress

Overexpression of wild-type human APP in mice causes cognitive déficits and pathological features unrelated to Abeta levels

Transgenic mice expressing mutant human amyloid precursor protein (APP) develop an age-dependent amyloid pathology and memory deficits, but no overt neuronal loss. Here, in mice overexpressing wild-type human APP (hAPPwt) we found an early memory impairment, particularly in the water maze and to a lesser extent in the object recognition task, but β-amyloid peptide (Aβ42) was barely detectable in the hippocampus. In these mice, hAPP processing was basically non-amyloidogenic, with high levels of APP carboxy-terminal fragments, C83 and APP intracellular domain. A tau pathology with an early increase in the levels of phosphorylated tau in the hippocampus, a likely consequence of enhanced ERK1/2 activation, was also observed. Furthermore, these mice presented a loss of synapse-associated proteins: PSD95, AMPA and NMDA receptor subunits and phosphorylated CaMKII. Importantly, signs of neurodegeneration were found in the hippocampal CA1 subfield and in the entorhinal cortex that were associated to a marked loss of MAP2 immunoreactivity. Conversely, in mice expressing mutant hAPP, high levels of Aβ42 were found in the hippocampus, but no signs of neurodegeneration were apparent. The results support the notion of Aβ- independent pathogenic pathways in Alzheimer's disease