Isidre Ferrer

Neuropathology and Pathogenesis of Encephalitis following Amyloid β Immunization in Alzheimer's Disease

Immunizing transgenic PDAPP mice, which overexpress mutant APP and develop β‐amyloid deposition resembling plaques in Alzheimers disease (AD), results in a decrease of amyloid burden when compared with non treated transgenic animals im‐munization with amyloid β peptide has been initiated in a randomised pilot study in AD. Yet a minority of patients developed a neurological complication consistent with meningoencephalitis and one patient died; the trial has been stopped. Neuropathological examination in that patient showed meningoencephalitis and focal atypically low numbers of diffuse and neuritic plaques but not of vascular amyloid nor regression of tau pathology in neurofibrillary tangles and neuropil threads. The present neuropathological study reports the second case of menigoencephalitis following immunization with amyloid‐β peptide in AD, and has been directed toward exploring mechanisms underlying decreased tau pathology in relation‐ with amyliod deposit regression, and possible molecular bases involved in the inflammatory response following immunization. Inflammatory infiltrates were composed of CD8+, CD3+, CD5+ and, rarely, CD7+ lymphocytes, whereas B lymphocytes and T cytotoxic cells CD16, CD57, TIA and graenzyme were negative. Characteristic neuropathological findings were focal depletion of diffuse and neuritic plaques, but not of amyloid angiopathy, and the presence of small numbers of extremely dense(collapsed) plaques surrounded by active microglia, and multinucleated giant cells filled with dense Aβ42and Aβ40, in addition to severe small cerebral blood Reduced amyloid burden was accompanied by low amyloid‐associated oxidative stress responses (reduced superoxide dismutase‐1:SOD‐1 expression) and by local inhibition of the stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK) and p38 kinase which are involved in tau phosphorylation. These results support the amyloid cascade of tau phosphorylation in AD regarding phosphorylation of tau in neurofibrillary tangles and β‐amyloid deposition in neuritic plaques, but not of tau in neurofibrillary tangles and threads. Furthermore, amyloid reduction was accompanied by increased expression of the PA28α/β inductor, and of LMP7, LMP2 and MECL1 subunits of the immunopro‐teasome in microglial and inflammatory cells surrounding collapsed plaques, and in multinucleated giant cells.Immunoproteasome subunit expression was accompanied by local presentation of MHC class molecules. Release of antigenic peptides derived from β‐amyloid processing may enhance T‐cell inflammatory responses accounting for the meningoencephalitis following amyloid‐β peptide immunization

Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat

Most of the mammalian genome is transcribed. This generates a vast repertoire of transcripts that includes protein-coding messenger RNAs, long non-coding RNAs (lncRNAs) and repetitive sequences, such as SINEs (short interspersed nuclear elements). A large percentage of ncRNAs are nuclear-enriched with unknown function. Antisense lncRNAs may form sense–antisense pairs by pairing with a protein-coding gene on the opposite strand to regulate epigenetic silencing, transcription and mRNA stability. Here we identify a nuclear-enriched lncRNA antisense to mouse ubiquitin carboxy-terminal hydrolase L1 (Uchl1), a gene involved in brain function and neurodegenerative diseases. Antisense Uchl1 increases UCHL1 protein synthesis at a post-transcriptional level, hereby identifying a new functional class of lncRNAs. Antisense Uchl1 activity depends on the presence of a 5′ overlapping sequence and an embedded inverted SINEB2 element. These features are shared by other natural antisense transcripts and can confer regulatory activity to an artificial antisense to green fluorescent protein. Antisense Uchl1 function is under the control of stress signalling pathways, as mTORC1 inhibition by rapamycin causes an increase in UCHL1 protein that is associated to the shuttling of antisense Uchl1 RNA from the nucleus to the cytoplasm. Antisense Uchl1 RNA is then required for the association of the overlapping sense protein-coding mRNA to active polysomes for translation. These data reveal another layer of gene expression control at the post-transcriptional level.

MicroRNA profiling of Parkinson’s disease brains identifies early downregulation of miR-34b/c which modulate mitochondrial function

MicroRNAs (miRNAs) are post-transcriptional gene expression regulators, playing key roles in neuronal development, plasticity and disease. Parkinsons disease (PD) is the second most common neurodegenerative disorder, characterized by the presence of protein inclusions or Lewy bodies and a progressive loss of dopaminergic neurons in the midbrain. Here, we have evaluated miRNA expression deregulation in PD brain samples. MiRNA expression profiling revealed decreased expression of miR-34b and miR-34c in brain areas with variable neuropathological affectation at clinical (motor) stages (Braak stages 4 and 5) of the disease, including the amygdala, frontal cortex, substantia nigra and cerebellum. Furthermore, misregulation of miR-34b/c was detected in pre-motor stages (stages 1-3) of the disease, and thus in cases that did not receive any PD-related treatment during life. Depletion of miR-34b or miR-34c in differentiated SH-SY5Y dopaminergic neuronal cells resulted in a moderate reduction in cell viability that was accompanied by altered mitochondrial function and dynamics, oxidative stress and reduction in total cellular adenosin triphosphate content. MiR-34b/c downregulation was coupled to a decrease in the expression of DJ1 and Parkin, two proteins associated to familial forms of PD that also have a role in idiopathic cases. Accordingly, DJ1 and Parkin expression was reduced in PD brain samples displaying strong miR-34b/c downregulation. We propose that early deregulation of miR-34b/c in PD triggers downstream transcriptome alterations underlying mitochondrial dysfunction and oxidative stress, which ultimately compromise cell viability. A better understanding of the cellular pathways controlling and/or controlled by miR-34b/c should allow identification of targets for development of therapeutic approaches.

Proteins in Human Brain Cortex Are Modified by Oxidation, Glycoxidation, and Lipoxidation EFFECTS OF ALZHEIMER DISEASE AND IDENTIFICATION OF LIPOXIDATION TARGETS

Diverse oxidative pathways, such as direct oxidation of amino acids, glycoxidation, and lipoxidation could contribute to Alzheimer disease pathogenesis. A global survey for the amount of structurally characterized probes for these reactions is lacking and could overcome the lack of specificity derived from measurement of 2,4-dinitrophenylhydrazine reactive carbonyls. Consequently we analyzed (i) the presence and concentrations of glutamic and aminoadipic semialdehydes, Nϵ-(carboxymethyl)-lysine, Nϵ-(carboxyethyl)-lysine, and Nϵ-(malondialdehyde)-lysine by means of gas chromatography/mass spectrometry, (ii) the biological response through expression of the receptor for advanced glycation end products, (iii) the fatty acid composition in brain samples from Alzheimer disease patients and agematched controls, and (iv) the targets of Nϵ-(malondialdehyde)-lysine formation in brain cortex by proteomic techniques. Alzheimer disease was associated with significant, although heterogeneous, increases in the concentrations of all evaluated markers. Alzheimer disease samples presented increases in expression of the receptor for advanced glycation end products with high molecular heterogeneity. Samples from Alzheimer disease patients also showed content of docosahexaenoic acid, which increased lipid peroxidizability. In accordance, Nϵ-(malondialdehyde)-lysine formation targeted important proteins for both glial and neuronal homeostasis such as neurofilament L, α-tubulin, glial fibrillary acidic protein, ubiquinol-cytochrome c reductase complex protein I, and the β chain of ATP synthase. These data support an important role for lipid peroxidation-derived protein modifications in Alzheimer disease pathogenesis.

A DNA methylation fingerprint of 1628 human samples

Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG-island hypermethylation and a loss of CpG methylation in non-CpG-island promoters. Although transformed cells are those in which DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the DNA methylation fingerprints obtained might be useful for translational purposes by showing that we are able to identify the tumor type origin of cancers of unknown primary origin (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues, and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases.

Current Advances on Different Kinases Involved in Tau Phosphorylation, and Implications in Alzheimers Disease and Tauopathies

Hyperphosphorylation and accumulation of tau in neurons (and glial cells) is one the main pathologic hallmarks in Alzheimers disease (AD) and other tauopathies, including Picks disease (PiD), progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease and familial frontotemporal dementia and parkinsonism linked to chromosome 17 due to mutations in the tau gene (FTDP-17-tau). Hyperphosphorylation of tau is regulated by several kinases that phosphorylate specific sites of tau in vitro. GSK-3-immunoprecipitated sarcosyl-insoluble fractions in AD have the capacity to phosphorylate recombinant tau. In addition, GSK-3 phosphorylated at Ser9, that inactivates GSK-3, is found in the majority of neurons with neurofibrillary tangles and dystrophic neurites of senile plaques in AD, and in Pick bodies and other phospho-tau-containing neurons and glial cells in other tauopathies. Increased expression of active kinases, including stress-activated kinase, c-Jun N-terminal kinase (SAPK/JNK) and kinase p38 has been found in brain homogenates in all the tauopathies. Strong active SAPK/JNK and p38 immunoreactivity has been observed restricted to neurons and glial cells containing hyperphosphorylated tau, as well as in dystrophic neurites of senile plaques in AD. Moreover, SAPK/JNK- and p38-immunoprecipitated sub-cellular fractions enriched in abnormal hyperphosphorylated tau have the capacity to phosphorylate recombinant tau and c-Jun and ATF-2 which are specific substrates of SAPK/JNK and p38 in AD and PiD. Interestingly, increased expression of phosphorylated (active) SAPK/JNK and p38 and hyperphosphorylated tau containing neurites have been observed around betaA4 amyloid deposits in the brain of transgenic mice (Tg 2576) carrying the double APP Swedish mutation. These findings suggest that betaA4 amyloid has the capacity to trigger the activation of stress kinases which, in turn, phosphorylate tau in neurites surrounding amyloid deposits. Complementary findings have been reported from the autopsy of two AD patients who participated in an amyloid-beta immunization trial and died during the course of immunization-induced encephalitis. The neuropathological examination of the brain showed massive focal reduction of amyloid plaques but not of neurofibrillary degeneration. Activation of SAPK/JNK and p38 were reduced together with decreased tau hyperphosphorylation of aberrant neurites in association with decreased amyloid plaques in both Tg2576 mice and human brains. These findings support the amyloid cascade hypothesis of tau phosphorylation mediated by stress kinases in dystrophic neurites of senile plaques but not that of neurofibrillary tangles and neuropil threads in AD.

A myriad of miRNA variants in control and Huntington’s disease brain regions detected by massively parallel sequencing

Huntington disease (HD) is a neurodegenerative disorder that predominantly affects neurons of the forebrain. We have applied the Illumina massively parallel sequencing to deeply analyze the small RNA populations of two different forebrain areas, the frontal cortex (FC) and the striatum (ST) of healthy individuals and individuals with HD. More than 80% of the small-RNAs were annotated as microRNAs (miRNAs) in all samples. Deep sequencing revealed length and sequence heterogeneity (IsomiRs) for the vast majority of miRNAs. Around 80–90% of the miRNAs presented modifications in the 3′-terminus mainly in the form of trimming and/or as nucleotide addition variants, while the 5′-terminus of the miRNAs was specially protected from changes. Expression profiling showed strong miRNA and isomiR expression deregulation in HD, most being common to both FC and ST. The analysis of the upstream regulatory regions in co-regulated miRNAs suggests a role for RE1-Silencing Transcription Factor (REST) and P53 in miRNAs downregulation in HD. The putative targets of deregulated miRNAs and seed-region IsomiRs strongly suggest that their altered expression contributes to the aberrant gene expression in HD. Our results show that miRNA variability is a ubiquitous phenomenon in the adult human brain, which may influence gene expression in physiological and pathological conditions.

Phosphorylated Map Kinase (ERK1, ERK2) Expression is Associated with Early Tau Deposition in Neurones and Glial Cells, but not with Increased Nuclear DNA Vulnerability and Cell Death, in Alzheimer Disease, Pick's Disease, Progressive Supranuclear Palsy and Corticobasal Degeneration

Abnormal tau phosphorylation and deposition in neurones and glial cells is one of the major features in tau pathies. The present study examines the involvement of the Ras/MEK/ERK pathway of tau phosphorylation in Alzheimer disease (AD), Picks disease (PiD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), by Western blotting, single and double‐labelling immunohistochemistry, and p21Ras activation assay. Since this pathway is also activated in several paradigms of cell death and cell survival, activated ERK expression is also analysed with double‐labelling immunohistochemistry and in situ end‐labelling of nuclear DNA fragmentation to visualise activated ERK in cells with increased nuclear DNA vulnerability. The MEK1 antibody recognises one band of 45 kD that identifies phosphorylation‐independent MEK1, whose expression levels are not modified in diseased brains. The ERK antibody recognises one band of 42 kD corresponding to the molecular weight of phosphorylation‐independent ERK2; the expression levels, as well as the immunoreactivity of ERK in individual cells, is not changed in AD, PiD, PSP and CBD. The antibody MAPK‐P distinguishes two bands of 44 kD and 42 kD that detect phosphorylated ERK1 and ERK2. MAPK‐P expression levels, as seen with Western blotting, are markedly increased in AD, PiD, PSP and CBD. Moreover, immunohistochemistry discloses granular precipitates in the cytoplasm of neurones in AD, mainly in a subpopulation of neurones exhibiting early tau deposition, whereas neurones with developed neurofibrillary tangles are less commonly immunostained. MAPK‐P also decorates neurones with Pick bodies in PiD, early tau deposition in neurones in PSP and CBD, and cortical achromatic neurones in CBD. In addition, strong MAPK‐P immunoreactivity is found in large numbers of tau‐positive glial cells in PSP and CBD, as seen with double‐labelling immunohistochemistry. Yet no co‐localisation of enhanced phosphorylated ERK immunoreactivity and nuclear DNA fragmentation is found in AD, PiD, PSP and CBD. Finally, activated Ras expression levels are increased in AD cases when compared with controls. These results demonstrate increased phosphorylated (active) ERK expression in association with early tau deposition in neurones and glial cells in taupathies, and suggest activated Ras as the upstream activator of the MEK/ERK pathway of tau phosphorylation in AD.

Dopamine induces autophagic cell death and α-synuclein increase in human neuroblastoma SH-SY5Y cells

Free cytoplasmic dopamine may be involved in the genesis of neuronal degeneration in Parkinsons disease and other such diseases. We used SH‐SY5Y human neuroblastoma cells to study the effect of dopamine on cell death, activation of stress‐induced pathways, and expression of α‐synuclein, the characteristic protein accumulated in Lewy bodies. We show that 100 and 500 μM dopamine causes a 40% and 60% decrease of viability, respectively, and triggers autophagy after 24 hr of exposure, characterized by the presence of numerous cytoplasmic vacuoles with inclusions. Dopamine causes mitochondrial aggregation in adherent cells prior to the loss of functionality. Plasma membrane and nucleus also maintain their integrity. Cell viability is protected by the dopamine transporter blocker nomifensine and the antioxidants N‐acetylcysteine and ascorbic acid. Dopamine activates the stress‐response kinases, SAPK/JNK and p38, but not ERK/MAPK or MEK, and increases α‐synuclein expression. Both cell viability and the increase in α‐synuclein expression are prevented by antioxidants; by the specific inhibitors of p38 and SAPK/JNK, SB203580 and SP600125, respectively; and by the inhibitor of autophagy 3‐methyladenine. This indicates that oxidative stress, stress‐activated kinases, and factors involved in autophagy up‐regulate α‐synuclein content. The results show that nonapoptotic death pathways are triggered by dopamine, leading to autophagy. These findings should be taken into account in the search for strategies to protect dopaminergic neurons from degeneration.

Down's dyndrome and Alzheimer's disease: dendritic spine counts in the hippocampus

SummarySamples of the hippocampus of four patients with Downs syndrome [two men aged 35 and 36 years with no evidence of Alzheimers disease (AD) and two patients aged 47 and 55 years with associated AD] were obtained at post mortem and processed according to the rapid Golgi method. A significant reduction in the number of dendritic spines (DS) was found in the apical (middle, distal and oblique segments) and basilar (thick and thin segments) dendritic arbors of CA1 and CA2–3 pyramidal neurons in patients with Downs syndrome and no AD when compared to age-matched controls. An additional decrease of DS in every segment occurred in Downs patients with associated AD when compared to agematched controls and Downs patients with no AD. In Downs syndrome (either associated or not to AD) thin basilar dendrites were the most severely involved; in AD patients CA1 pyramids were more severely affected than pyramidal neurons of the CA2–3 subfield.