Margarita Carmona

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.

Phosphorylated mitogen-activated protein kinase (MAPK/ERK-P), protein kinase of 38 kDa (p38-P), stress-activated protein kinase (SAPK/JNK-P), and calcium/calmodulin-dependent kinase II (CaM kinase II) are differentially expressed in tau deposits in neurons and glial cells in tauopathies.

Summary. Calcium/calmodulin-dependent kinase II (α- and β-CaM kinase II), and phosphorylated mitogen-activated extracellular signal-regulated protein kinase (MAPK/ERK-P), phosphorylated protein kinase of 38 kDa (p38-P) and phosphorylated stress-activated protein kinase (SAPK/JNK-P) expression have been examined in Alzheimer disease (AD), Picks disease (PiD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). The study was carried out to increase understanding of the signals that may regulate tau phosphorylation in tauopathies. MAPK/ERK-P was found in a subset of neurons and glial cells bearing abnormal tau deposition, but rarely in neurofibrillary tangles. Strong p38-P immunoreactivity was observed in about 50–70% of neurons with neurofibrillary tangles and in dystrophic neurites of senile plaques in AD. Strong p38-P immunoreactivity was seen in practically all Pick bodies in PiD, and in most neurons with neurofibrillary degeneration or with tau deposits (pre-tangle neurons) in PSP and CBD, as revealed with single and double-labeling immunohistochemistry to p38-P and tau. In addition, strong p38-P immunoreactivity was present in tau-positive astrocytes and in coiled bodies in PSP and CBD. Single and double-labeling immunohistochemistry to MAPK/ERK-P and p38-P disclosed that MAPK/ERK-P appeared at early stages of tau phosphorylation in neurons and glial cells in tauopathies, and that MAPK/ERK-P and p38-P co-localize only in a subset of neurons and glial cells with phosphorylated tau deposits. SAPK/JNK-P immunoreactivity was seen in a subset of neurons, including many neurons with neurofibrillary degeneration, and in glial cells accumulating abnormal tau, in AD, PiD, PSP and CBD. Double-labeling immunohistochemistry disclosed partial co-localization of SAPK/JNK-P and either MAPK/ERK-P or p-38-P immunoreactivity. These findings indicate that MAPK/ERK-P, SAPK/JNK-P and p-38-P are differentially expressed in association with tau deposits in tauopathies. Finally, CaM kinase II is present in neurons but not in glial cells, thus suggesting no role of CaM kinase II in tau phosphorylation of glial cells. These observations, together with previous results of in vitro studies, support the idea that several MAPK/ERK, SAPK/JNK, p38 and CaM kinase II may participate in tau phosphorylation in tauopathies. Lack of co-localization between MAPK/ERK-P, SAPK/JNK-P and p-38-P over-expression, and staining with the method of in situ end-labeling of nuclear DNA fragmentation in individual cells indicate that over-expression of these kinases is not linked with increased nuclear DNA vulnerability in AD, PiD, PSP and CBD.

Active, phosphorylation-dependent mitogen-activated protein kinase (MAPK/ERK), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p38 kinase expression in Parkinson's disease and Dementia with Lewy bodies

Summary. The expression of mitogen-activated protein kinases, extracellular signal-regulated kinases (MAPK/ERK), stress-activated protein kinases, c-Jun N-terminal kinases (SAPK/JNK), and p38 kinases is examined in Parkinson disease (PD), in Dementia with Lewy bodies (DLB), covering common and pure forms, and in age-matched controls. The study is geared to gaining understanding about the involvement of these kinases in the pathogenesis of Lewy bodies (LBs) and associated tau deposits in Alzheimer changes in the common form of DLB. Active, phosphorylation dependent MAPK (MAPK-P) is found as granular cytoplasmic inclusions in a subset of cortical neurons bearing abnormal tau deposits in common forms of DLB. Phosphorylated p-38 (p-38-P) decorates neurons with neurofibrillary tangles and dystrophic neurites of senile plaques in common forms of DLB. Phosphorylated SAPK/JNK (SAPK/JNK-P) expression occurs in cortical neurons with neurofibrillary tangles in the common form of DLB. Lewy bodies (LBs) in the brain stem of PD and DLB are stained with anti-ERK-2 antibodies, but they are not recognized by MAPK-P, SAPK/JNK-P and p-38-P. Yet MAPK-P, p-38-P and SAPK/JNK-P immunoreactivity is found in cytoplasmic granules in the vicinity of LBs or in association with irregular-shaped or diffuse α-synuclein deposits in a small percentage of neurons, not containing phosphorylated tau, of the brain stem in PD and DLB. MAPK-P, p-38-P and SAPK-P are not expressed in cortical LBs or in cortical neurons with α-synuclein-only inclusions in DLB. MAPK-P, p-38-P and SAPK/JNK-P are not expressed in α-synuclein-positive neurites (Lewy neurites) in PD and DLB as revealed by double-labeling immunohistochemistry. These results show that MAPKs are differentially regulated in neurons with α-synuclein-related inclusions and in neurons with abnormal tau deposits in DLB. Moreover, different kinase expression in brain stem and cortical LBs suggest a pathogenesis of brain stem and cortical LBs in LB diseases. Finally, no relationship has been observed between MAPK-P, p-38-P and SAPK/JNK-P expression and increased nuclear DNA vulnerability, as revealed with the method of in situ end-labeling of nuclear DNA fragmentation, and active, cleaved caspase-3 expression in neurons and glial cells in the substantia nigra in PD and DLB.

Brain protein preservation largely depends on the postmortem storage temperature: implications for study of proteins in human neurologic diseases and management of brain banks: a BrainNet Europe Study

The present study was designed to reveal protein modifications in control cases related with postmortem delay and temperature of storage in 3 paradigms in which the same postmortem tissue sample (frontal cortex) was frozen a short time after death or stored at 1°C, 4°C, or room temperature and then frozen at −80°C at different intervals. No evidence of protein degradation as revealed with monodimensional gel electrophoresis and Western blotting was observed in samples artificially stored at 1°C and then frozen at different intervals up to 50 hours after death. However, the levels of several proteins were modified in samples stored at 4°C and this effect was more marked in samples stored at room temperature. Two-dimensional gel electrophoresis and mass spectrometry further corroborated these observations and permitted the identification of other proteins vulnerable or resistant to postmortem delay. Finally, gel electrophoresis and Western blotting of sarkosyl-insoluble fractions in Alzheimer disease showed reduced intensity of phospho-tau-specific bands with postmortem delay with the effects being more dramatic when the brain samples were stored at room temperature for long periods. These results emphasize the necessity of reducing the body temperature after death to minimize protein degradation.

Neuropathology of sporadic Parkinson disease before the appearance of parkinsonism: preclinical Parkinson disease

Parkinson disease (PD) is no longer considered a complex motor disorder characterized by parkinsonism but rather a systemic disease with variegated non-motor deficits and neurological symptoms, including impaired olfaction, sleep disorders, gastrointestinal and urinary abnormalities and cardiovascular dysfunction, in addition to other symptoms and signs such as pain, depression and mood disorders. Many of these alterations appear before or in parallel with motor deficits and then worsen with disease progression. Although there is a close relation between motor symptoms and the presence of Lewy bodies (LBs) and neurites filled with abnormal α-synuclein, other neurological alterations are independent of LBs, thereby indicating that different mechanisms probably converge in the degenerative process. This review presents cardinal observations at very early stages of PD and provides personal experience based on the study of a consecutive series of brains with PD-related pathology and without parkinsonism, mainly cases categorized as stages 2–3 of Braak. Alterations in the substantia nigra, striatum and frontal cortex in pPD are here revised in detail. Early modifications in the substantia nigra at pre-motor stages of PD (preclinical PD: pPD) include abnormal small aggregates of α-synuclein which is phosphorylated, nitrated and oxidized, and which exhibits abnormal solubility and truncation. This occurs in association with a plethora of altered molecular events including increased oxidative stress, altered oxidative stress responses, altered balance of L-ferritin and H-ferritin, reduced expression of neuronal globin α and β chains in neurons with α-synuclein deposits, increased expression of endoplasmic reticulum stress markers, increased p62 and ubiquitin immunoreactivity in relation to α-synuclein deposits, and altered distribution of LC3 and other autophagosome/lysosome markers. In spite of the relatively small decrease in the number of dopaminergic neurons in the substantia nigra, which does not reach thresholds causative of parkinsonism, levels of tyrosine hydroxylase and cannabinoid 1 receptor are reduced, whereas levels of adenosine receptor 2A are increased in the caudate in pPD. Moreover, biochemical alterations are also present in the cerebral cortex (at least in the frontal cortex) in pPD including increased oxidative stress and oxidative damage to proteins α-synuclein, β-synuclein, superoxide dismutase 2, aldolase A, enolase 1, and glyceraldehyde dehydrogenase, among others, indicating post-translational modifications of PD-related proteins, and suggesting altered function of pathways involved in glycolysis and energy metabolism in the cerebral cortex in pPD. Current evidence suggests convergence of several altered metabolic pathways leading to chronic neuronal dysfunction, mainly manifested as sub-optimal energy metabolism, altered synaptic function, oxidative and endoplasmic reticulum stress damage and corresponding altered responses, among others. By understanding that these alterations occur at very early stages of PD and that neuronal fatigue and exhaustion may precede, for years, cell death and neuronal loss, we may direct therapeutic strategies towards the prevention and delay of disease progression starting at pre-parkinsonian stages of PD.

Transforming growth factor-α (TGF-α) and epidermal growth factor-receptor (EGF-R) immunoreactivity in normal and pathologic brain

Abstract Transforming growth factor α (TGF-α) and epidermal growth factor-receptor (EGF-R) immunoreactivity is observed in the majority of neurons, and in maturing astrocytes, in the developing and adult brain of humans and different species of animals. TGF-α and EGF-R co-localize in most neurons and maturing astrocytes, suggesting that most TGF-α producing cells are EGF-R-expressing cells. TGF-α and EGF-R immunoreactivity decrease in damaged areas following different insults. However, EGF-R appears in reactive glia, mostly reactive astrocytes, within and surrounding the damaged areas. TGF-α and EGF-R immunoreactivity is found in neurons of patients affected by Alzheimers disease and other forms of dementia, and in neurons of patients suffering from epilepsy owing to different causes, thus pointing to the conclusion that TGF-α does not play a significant role in these pathologies. However, EGF-R immunoreactivity occurs in reactive astrocytes and microglia in subacute but not chronic lesions in human cases. Since TGF-α is a membrane-anchored growth factor, which may be cleaved leading to the formation of soluble forms, and both the membrane-anchored and soluble forms have the capacity to activate the EGF-R, it is feasible that TGF-α in the nervous system may act upon EGF-R-containing neurons through different mechanisms. In addition to distant effects resulting from the release of soluble TGF-α, local effects may be produced by establishing direct cell-to-cell contacts (juxtacrine stimulation), or in cells expressing both TGF-α and EGF-R (autocrine stimulation).

Prion protein expression in senile plaques in Alzheimer's disease

Abstract. Prion protein (PrPC) is a glycolipid-anchored cell membrane sialoglycoprotein that localises in presynaptic membranes. Since synapses are vulnerable to Alzheimers disease (AD), the present study examines PrPC expression in senile plaques, one of the major structural abnormalities in AD, by single- and double-labelling immunohistochemistry. Punctate PrPC immunoreactivity is found in diffuse plaques, whereas isolated large coarse PrPC-positive granules reminiscent of dystrophic neurites are observed in neuritic plaques. Finally, PrPC deposition also occurs as dense filamentous and amorphous precipitates in amyloid cores of senile plaques, but not in the walls of blood vessels with amyloid angiopathy. In contrast to PrPC, βA4-amyloid immunoreactivity is preserved and even enhanced following incubation of the tissue sections with proteinase K prior to immunohistochemistry, thus indicating no PrPC and βA4-amyloid cross-reactivity in dense amyloid cores of senile plaques. Punctate PrPC deposition in diffuse plaques is similar to that of synaptophysin, a synaptic vesicle-associated protein, as already reported in other studies. Immunoprecipitation, electrophoresis and Western blot studies have shown that synaptophysin, amyloid precursor protein (APP) and βA4 do not co-precipitate with PrP. These results suggest that synaptophysin, APP and βA4 are likely not bound to PrP. PrPC accumulation in βA4-amyloid dense cores may be the consequence of the release of PrP into the extracellular space. Whether PrPC accumulation in the extracellular space is the result of impaired endocytosis and subsequent hydrolysis in the endosomal compartment, in contrast to normal degradation of PrPC, resulting from or occurring in parallel to abnormal APP degradation, deserves further study.

Glial and neuronal tau pathology in tauopathies: characterization of disease-specific phenotypes and tau pathology progression.

Tauopathies are degenerative diseases characterized by the accumulation of phosphorylated tau in neurons and glial cells. With some exceptions, tau deposits in neurons are mainly manifested as pretangles and tangles unrelated to the tauopathy. It is thought that abnormal tau deposition in neurons occurs following specific steps, but little is known about the progression of tau pathology in glial cells in tauopathies. We compared tau pathology in different astrocyte phenotypes and oligodendroglial inclusions with that in neurons in a large series of tauopathies, including progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, Pick disease, frontotemporal lobar degenerations (FTLD) associated with mutations in the tau gene, globular glial tauopathy (GGT), and tauopathy in the elderly. Our findings indicate that disease-specific astroglial phenotypes depend on i) the primary amino acid sequence of tau (mutated tau, 3Rtau, and 4Rtau); ii) phospho-specific sites of tau phosphorylation, tau conformation, tau truncation, and ubiquitination in that order (which parallel tau modifications related to pretangle and tangle stages in neurons); and iii) modifications of the astroglial cytoskeleton. In contrast to astrocytes, coiled bodies in oligodendrocytes have similar characteristics whatever the tauopathy, except glial globular inclusions in GGT, and coiled bodies and globular oligodendroglial inclusions in FTLD-tau/K317M. These observations indicate that tau pathology in glial cells largely parallels, but is not identical to, that in neurons in many tauopathies.

Amyloid Generation and Dysfunctional Immunoproteasome Activation with Disease Progression in Animal Model of Familial Alzheimer's Disease

Double‐transgenic amyloid precursor protein/presenilin 1 (APP/PS1) mice express a chimeric mouse/human APP bearing the Swedish mutation (Mo/HuAPP695swe) and a mutant human PS1‐dE9 both causative of familial Alzheimers disease (FAD). Transgenic mice show impaired memory and learning performance from the age of 6 months onwards. Double‐transgenic APP/PS1 mice express altered APP and PS1 mRNAs and proteins, reduced β‐secretase 1 (BACE1) mRNA and normal BACE1 protein, all of which suggest a particular mechanism of amyloidogenesis when compared with sporadic AD. The first β‐amyloid plaques in APP/PS1 mice appear at 3 months, and they increase in number and distribution with disease progression in parallel with increased levels of brain soluble β‐amyloid 1–42 and 1–40, but also with reduced 1–42/1–40 ratio with age. Amyloid deposition in plaques is accompanied by altered mitochondria and increased oxidative damage, post‐translational modifications and accumulation of altered proteins at the dystrophic neurites surrounding plaques. Degradation pathways are also modified with disease progression including activation of the immunoproteasome together with variable alterations of the different protease activities of the ubiquitin‐proteasome system. Present observations show modifications in the production of β‐amyloid and activation and malfunction of the subcellular degradation pathways that have general implications in the pathogenesis of AD and more particularly in specificities of FAD amyloidogenesis.

Neuroinflammatory signals in alzheimer disease and APP/PS1 transgenic mice: Correlations with plaques, tangles, and oligomeric species

Abstract To understand neuroinflammation-related gene regulation during normal aging and in sporadic Alzheimer disease (sAD), we performed functional genomics analysis and analyzed messenger RNA (mRNA) expression by quantitative reverse transcription–polymerase chain reaction of 22 genes involved in neuroinflammation-like responses in the cerebral cortex of wild-type and APP/PS1 transgenic mice. For direct comparisons, mRNA expression of 18 of the same genes was then analyzed in the entorhinal cortex, orbitofrontal cortex, and frontal cortex area 8 of middle-aged human subjects lacking Alzheimer disease–related pathology and in older subjects with sAD pathology covering Stages I–II/0(A), III–IV/A–B, and V–VI/C of Braak and Braak classification. Modifications of cytokine and immune mediator mRNA expression were found with normal aging in wild-type mice and in middle-aged individuals and patients with early stages of sAD-related pathology; these were accompanied by increased protein expression of certain mediators in ramified microglia. In APP/PS1 mice, inflammatory changes coincided with &bgr;-amyloid (A&bgr;) deposition; increased levels of soluble oligomers paralleled the modified mRNA expression of cytokines and mediators in wild-type mice. In patients with sAD, regulation was stage- and region-dependent and not merely acceleration and exacerbation of mRNA regulation with aging. Gene regulation at first stages of AD was not related to hyperphosphorylated tau deposition in neurofibrillary tangles, A&bgr; plaque burden, concentration of A&bgr;1–40 (A&bgr;40) and A&bgr;1–42 (A&bgr;42), or fibrillar A&bgr; linked to membranes but rather to increased levels of soluble oligomers. Thus, species differences and region- and stage-dependent inflammatory responses in sAD, particularly at the initial stages, indicate the need to identify new anti-inflammatory compounds with specific molecular therapeutic targets.