Modelling the neuropathology of lysosomal storage disorders through disease-specific human induced pluripotent stem cells.

Mucopolysaccharidosis II (MPS II) is a lysosomal storage disorder (LSD), caused by iduronate 2-sulphatase (IDS) enzyme dysfunction. The neuropathology of the disease is not well understood, although the neural symptoms are currently incurable. MPS II-patient derived iPSC lines were established and differentiated to neuronal lineage. The disease phenotype was confirmed by IDS enzyme and glycosaminoglycan assay. MPS II neuronal precursor cells (NPCs) showed significantly decreased self-renewal capacity, while their cortical neuronal differentiation potential was not affected. Major structural alterations in the ER and Golgi complex, accumulation of storage vacuoles, and increased apoptosis were observed both at protein expression and ultrastructural level in the MPS II neuronal cells, which was more pronounced in GFAP + astrocytes, with increased LAMP2 expression but unchanged in their RAB7 compartment. Based on these finding we hypothesize that lysosomal membrane protein (LMP) carrier vesicles have an initiating role in the formation of storage vacuoles leading to impaired lysosomal function. In conclusion, a novel human MPS II disease model was established for the first time which recapitulates the in vitro neuropathology of the disorder, providing novel information on the disease mechanism which allows better understanding of further lysosomal storage disorders and facilitates drug testing and gene therapy approaches.

Lysosomal response in relation to α-synuclein pathology differs between Parkinson's disease and multiple system atrophy.

Intracellular deposition of pathologically altered α-synuclein mostly in neurons characterises Parkinson's disease (PD), while its accumulation predominantly in oligodendrocytes is a feature of multiple system atrophy (MSA). Recently a prion-like spreading of pathologic α-synuclein has been suggested to play a role in the pathogenesis of PD and MSA. This implicates a role of protein processing systems, including lysosomes, supported also by genetic studies in PD. However, particularly for MSA, the mechanism of cell-to-cell propagation of α-synuclein is yet not fully understood. To evaluate the significance of lysosomal response, we systematically compared differently affected neuronal populations in PD, MSA, and non-diseased brains using morphometric immunohistochemistry (cathepsin D), double immunolabelling (cathepsin D/α-synuclein) laser confocal microscopy, and immunogold electron microscopy for the disease associated α-synuclein. We found that i) irrespective of the presence of neuronal inclusions, the volume density of cathepsin D immunoreactivity significantly increases in affected neurons of the pontine base in MSA brains; ii) volume density of cathepsin D immunoreactivity increases in nigral neurons in PD without inclusions and with non-ubiquitinated pre-aggregates of α-synuclein, but not in neurons with Lewy bodies; iii) cathepsin D immunoreactivity frequently colocalises with α-synuclein pre-aggregates in nigral neurons in PD; iv) ultrastructural observations confirm disease-associated α-synuclein in neuronal and astrocytic lysosomes in PD; v) lysosome-associated α-synuclein is observed in astroglia and rarely in oligodendroglia and in neurons in MSA. Our observations support a crucial role for the neuronal endosomal-lysosomal system in the processing of α-synuclein in PD. We suggest a distinct contribution of lysosomes to the pathogenesis of MSA, including the possibility of oligodendroglial and eventually neuronal uptake of exogenous α-synuclein in MSA.

Intracellular processing of disease-associated α-synuclein in the human brain suggests prion-like cell-to-cell spread.

Dementia with Lewy bodies (DLB), Parkinson's disease (PD) and multiple system atrophy are characterized by the deposition of disease-associated α-synuclein. In the present study we 1) examined the molecular specificity of the novel anti-α-synuclein 5G4 antibody; 2) evaluated immunoreactivity patterns and their correlation in human brain tissue with micro- and astrogliosis in 57 cases with PD or DLB; and 3) performed a systematic immunoelectron microscopical mapping of subcellular localizations. 5G4 strongly binds to the high molecular weight fraction of ß-sheet rich oligomers, while no binding to primarily disordered oligomers or monomers was observed. We show novel localizations of disease-associated α-synuclein including perivascular macrophages, ependyma and cranial nerves. α-Synuclein immunoreactive neuropil dots and thin threads associate more with glial reaction than Lewy bodies alone. Astrocytic α-synuclein is an important component of the pathology. Furthermore, we document ultrastructurally the pathway of processing of disease-associated α-synuclein within neurons and astroglial cells. Interaction of mitochondria and disease-associated α-synuclein plays a key role in the molecular-structural cytopathogenesis of disorders with Lewy bodies. We conclude that 1) the 5G4 antibody has strong selectivity for ß-sheet rich α-synuclein oligomers; 2) Lewy bodies themselves are not the most relevant morphological substrate that evokes tissue lesioning; 3) both neurons and astrocytes internalize disease-associated α-synuclein in the human brain, suggesting prion-like cell-to-cell spread of α-synuclein by uptake from surrounding structures, as shown previously in experimental observations.

Voluntary exercise does not increase gastrointestinal motility but increases spatial memory, intestinal eNOS, Akt levels, and Bifidobacteria abundance in the microbiome.

The interaction between the gut and brain is a great puzzle since it is mediated by very complex mechanisms. Therefore, the possible interactions of the brain-exercise-intestine-microbiome axis were investigated in a control (C, N = 6) and voluntarily exercised (VE, N = 8) middle-aged rats. The endurance capacity was assessed by VO2max on the treadmill, spatial memory by the Morris maze test, gastrointestinal motility by EMG, the microbiome by 16S RNA gene amplicon sequencing, caveolae by electron microscopy, and biochemical assays were used to measure protein levels and production of reactive oxygen species (ROS). Eight weeks of voluntary running increased VO2max, and spatial memory was assessed by the Morris maze test but did not significantly change the motility of the gastrointestinal tract or production of ROS in the intestine. The protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) protein levels significantly increased in the intestine, while peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), mitochondrial transcription factor A (TFAM), nuclear respiratory factor 1 (NFR1), SIRT1, SIRT3, nicotinamide phosphoribosyl transferase (NAMPT), and nuclear factor κB (NF-κB) did not change. On the other hand, voluntary exercise increased the number of caveolae in the smooth muscles of the intestine and relative abundance of Bifidobacteria in the microbiome, which correlated with the Akt levels in the intestine. Voluntary exercise has systemic effects and the relationship between intestinal Akt and the microbiome of the gastrointestinal tract could be an important adaptive response.

Novel crystalloid oligodendrogliopathy in hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) comprises a group of clinically and genetically heterogeneous disorders associated with spastic paraparesis (pure HSP) with or without additional neurological symptoms (complicated HSP). Here we present a case of an adult-onset, apparently autosomal-dominant, complicated form of HSP. Onset of clinical symptoms was at the age 40 years and characterised by slowly progressive corticospinal tract dysfunction, dysarthria, disorientation, extrapyramidal symptoms, and bilateral ptosis. Cranial MRI revealed hyperintensities on T2-weighted sequences mostly in the posterior limb of the internal capsule. The proband deceased at the age of 64 years. As morphological substrate for the slowly progressive clinical symptoms, comprehensive neuropathological and ultrastructural evaluation revealed a novel oligodendrogliopathy with distinctive, partly ubiquitinated and p62 positive fibrillar inclusions evolving into crystalloid deposits, containing elements of the oligodendroglial cytoskeleton (α- and ß-tubulin, TPPP/p25). In the central nervous system, accumulation of crystalloid structures has been related to histiocytes but not to glial cells. This study has implications for the understanding on how the human central nervous system reacts to protracted dysfunction and disruption of the oligodendroglial cytoskeleton, including development of crystalloid structures, which have not yet been reported in neurodegenerative diseases including HSP.

A peculiar constellation of tau pathology defines a subset of dementia in the elderly.

Sporadic tauopathies are characterized by differential cellular and topographical predominance of phospho-tau immunoreactivity and biochemical distinction of the tau protein. Established entities include progressive supranuclear palsy, corticobasal degeneration, Pick's disease, and argyrophilic grain disease. During a community-based longitudinal study on aging, we detected tau pathologies not compatible with these categories. We immunostained for different phospho-tau epitopes, 4R and 3R tau isoforms, α-synuclein, amyloid-ß, and phospho-TDP-43, analyzed the MAPT and ApoE genes, and performed western blotting for the tau protein. The mean age of patients (4 women, 3 men) was 83.8 years. Clinical presentations combined dementia with psychiatric symptoms and/or parkinsonism. In addition to neurofibrillary tangles and diffuse neuronal cytoplasmic tau immunoreactivity, the neuropathology was characterized by peculiar cytopathologies (diffuse granular immunopositivity of astrocytic processes and patchy accumulation of thin threads) in a distinctive distribution (frontal and temporal cortices, hippocampus, amygdala, basal ganglia, locus coeruleus, and substantia nigra). Argyrophilic grains were detected in four patients. Few to moderate densities of neuritic plaques but widespread phospho-TDP-43 pathology was observed in five patients. There was variability in the H1/H2 and ApoE alleles and biochemical features of tau protein. We propose these cases as complex tauopathy with a characteristic constellation: some features of primary tauopathies and Alzheimer's disease mixed with additional cytopathologies including a distinctive astrogliopathy, in a characteristic distribution of lesions. These complex tauopathies in the elderly deserve specific diagnostic and eventually therapeutic considerations.

Genetic Creutzfeldt-Jakob disease associated with the E200K mutation: characterization of a complex proteinopathy.

The E200K mutation is the most frequent prion protein gene (PRNP) mutation detected worldwide that is associated with Creutzfeldt-Jakob disease (CJD) and thought to have overlapping features with sporadic CJD, yet detailed neuropathological studies have not been reported. In addition to the prion protein, deposition of tau, α-synuclein, and amyloid-ß has been reported in human prion disease. To describe the salient and concomitant neuropathological alterations, we performed a systematic clinical, neuropathological, and biochemical study of 39 individuals carrying the E200K PRNP mutation originating from different European countries. The most frequent clinical symptoms were dementia and ataxia followed by myoclonus and various combinations of further symptoms, including vertical gaze palsy and polyneuropathy. Neuropathological examination revealed relatively uniform anatomical pattern of tissue lesioning, predominating in the basal ganglia and thalamus, and also substantia nigra, while the deposition of disease-associated PrP was more influenced by the codon 129 constellation, including different or mixed types of PrP(res) detected by immunoblotting. Unique and prominent intraneuronal PrP deposition involving brainstem nuclei was also noted. Systematic examination of protein depositions revealed parenchymal amyloid-ß in 53.8%, amyloid angiopathy (Aß) in 23.1%, phospho-tau immunoreactive neuritic profiles in 92.3%, neurofibrillary degeneration in 38.4%, new types of tau pathology in 33.3%, and Lewy-type α-synuclein pathology in 15.4%. TDP-43 and FUS immunoreactive protein deposits were not observed. This is the first demonstration of intensified and combined neurodegeneration in a genetic prion disease due to a single point mutation, which might become an important model to decipher the molecular interplay between neurodegeneration-associated proteins.

Peppermint extract inhibits protein aggregation.

The extracts of 7 herbs were screened and compared for their functional ability to inhibit the aggregation of trypsin as an appropriate model protein for in vitro fibrillation in aqueous ethanol at pH 7.0. Turbidity measurements, total phenolic content determination, aggregation kinetics, Congo red binding assay as well as transmission electron microscopy were used to analyse the inhibition of amyloid fibril formation. This correlated with the total phenolic content of the herb extracts. The peppermint extract proved to be the most potent anti-amyloidogenic agent. Results showed that the peppermint extract exerted dose-dependent inhibitory effect on trypsin fibril formation.

Human Induced Pluripotent Stem Cell-Derived 3D-Neurospheres are Suitable for Neurotoxicity Screening.

We present a hiPSC-based 3D in vitro system suitable to test neurotoxicity (NT). Human iPSCs-derived 3D neurospheres grown in 96-well plate format were characterized timewise for 6-weeks. Changes in complexity and homogeneity were followed by immunocytochemistry and transmission electron microscopy. Transcriptional activity of major developmental, structural, and cell-type-specific markers was investigated at weekly intervals to present the differentiation of neurons, astrocytes, and oligodendrocytes. Neurospheres were exposed to different well-known toxicants with or without neurotoxic effect (e.g., paraquat, acrylamide, or ibuprofen) and examined at various stages of the differentiation with an ATP-based cell viability assay optimized for 3D-tissues. Concentration responses were investigated after acute (72 h) exposure. Moreover, the compound-specific effect of rotenone was investigated by a panel of ER-stress assay, TUNEL assay, immunocytochemistry, electron microscopy, and in 3D-spheroid based neurite outgrowth assay. The acute exposure to different classes of toxicants revealed distinct susceptibility profiles in a differentiation stage-dependent manner, indicating that hiPSC-based 3D in vitro neurosphere models could be used effectively to evaluate NT, and can be developed further to detect developmental neurotoxicity (DNT) and thus replace or complement the use of animal models in various basic research and pharmaceutical applications.

The Inhibitory Effect of Panax Ginseng Extract on Amyloid-like Fibril Formation of Trypsin in Aqueous Ethanol.

BACKGROUND: In case of several chronic diseases, prevention is could be more effective than treatment. Functional foods that contain significant amounts of bioactive components gained considerable attention not only in traditional but in modern medicine as well. We have investigated how P. ginseng extract inhibits the in vitro formation of amyloid-like fibrils of phenylmethylsulfonyl- trypsin (PMS-trypsin) in 60% ethanol at pH 7.0. The model system used is non-physiological, but it is capable of detecting the anti-amyloidogenic effect of the various agents. OBJECTIVE: The main objective of this study was to examine the possible inhibitory effect of ginseng extract on amyloid-like fibril formation of trypsin in aqueous ethanol. METHODS: The amyloid formation and aggregation kinetics of PMS-trypsin was studied by turbidity measurements, Congo Red (CR) binding assays, size exclusion chromatography and Electronic Circular Dichroism (ECD) measurements and the shapes of amyloid fibrils became visible by Transmission Electron Microscopy (TEM). RESULTS: In the presence of 500-fold diluted P. ginseng extract in the incubation mixture, the absorption at 350 nm decreased to 47.1% after incubation for 24 h, compared relative to the sample which contained no additives. CR binding experiments suggested that the aggregates in our samples have amyloid-like properties, and P. ginseng extract inhibits the amyloid-like fibril formation of PMS-trypsin depending on concentration. Our results show that the ginseng extract does not bind to the fibrils. In the absence of P. ginseng extracts large sized colloid aggregates were abundant. Adding P. ginseng extracts to our samples decreased the light dispersion of the solution. This is due to the decrease of the rate of the aggregation or to the smaller size of the aggregates evolved. Our results show that the presence of ginseng extract helps to maintain the native structure of the protein. In the presence of 500-fold diluted P. ginseng extract, TEM images demonstrated, that P. ginseng extract has inhibitory effect on the formation of amyloid-like fibrils of PMS-trypsin. CONCLUSION: The results indicated that P. ginseng extract significantly inhibits the formation of amyloid-like fibrils of PMS-trypsin in aqueous ethanol, and helps to maintain the native structure of the protein. The rate of inhibition depends on concentration. P. ginseng extract is an efficient antiamyloidogenic agent.

Filamentous Aggregation of Sequestosome-1/p62 in Brain Neurons and Neuroepithelial Cells upon Tyr-Cre-Mediated Deletion of the Autophagy Gene Atg7.

Defects in autophagy and the resulting deposition of protein aggregates have been implicated in aging and neurodegenerative diseases. While gene targeting in the mouse has facilitated the characterization of these processes in different types of neurons, potential roles of autophagy and accumulation of protein substrates in neuroepithelial cells have remained elusive. Here we report that Atg7f/f Tyr-Cre mice, in which autophagy-related 7 (Atg7) is conditionally deleted under the control of the tyrosinase promoter, are a model for accumulations of the autophagy adapter and substrate sequestosome-1/p62 in both neuronal and neuroepithelial cells. In the brain of Atg7f/f Tyr-Cre but not of fully autophagy competent control mice, p62 aggregates were present in sporadic neurons in the cortex and other brain regions as well in epithelial cells of the choroid plexus and the ependyma. Western blot analysis confirmed a dramatic increase of p62 abundance and formation of high-molecular weight species of p62 in the brain of Atg7f/f Tyr-Cre mice relative to Atg7f/f controls. Immuno-electron microscopy showed that p62 formed filamentous aggregates in neurons and ependymal cells. p62 aggregates were also highly abundant in the ciliary body in the eye. Atg7f/f Tyr-Cre mice reached an age of more than 2 years although neurological defects manifesting in abnormal hindlimb clasping reflexes were evident in old mice. These results show that p62 filaments form in response to impaired autophagy in vivo and suggest that Atg7f/f Tyr-Cre mice are a model useful to study the long-term effects of autophagy deficiency on the homeostasis of different neuroectoderm-derived cells.

[Human prion diseases: the Hungarian experience]. / Emberi prionbetegségek: magyarországi tapasztalatok.

BACKGROUND: Sporadic Creutzfeldt-Jakob disease is the most frequent human prion disease. Genetic forms are associated with mutations in the human prion protein gene (PRNP) and thought to comprise 5-15% of cases. Acquired forms include iatrogenic and variant Creutzfeldt-Jakob disease. The latter is associated with the bovine spongiform encephalopathy. We recently reported the high incidence of genetic Creutzfeldt-Jakob disease in Hungary. MATERIALS AND METHODS: In the present study we summarize the results of a widened investigation comprising Creutzfeldt-Jakob disease cases collected in the National Institute of Psychiatry and Neurology, Hungary in the last 12 years. We examined the disease forms and their geographical distribution. RESULTS: Our study involved 155 patients. The four major results are as follows: 1. In Hungary we detected only sporadic and genetic forms of human prion disease, while iatrogenic and variant Creutzfeldt-Jakob disease were not observed. 2. The proportion of genetic prion disease (E200K mutation), similarly to Slovakia, is higher than reported worldwide. Our observations indicate that at least every third case is genetic Creutzfeldt-Jakob disease. The mean incidence of genetic Creutzfeldt-Jakob disease (0.42/million) is unusually high. Especially the year 2006 was striking when the incidence of genetic Creutzfeldt-Jakob disease was 1.4/million. 3. More than half of genetic Creutzfeldt-Jakob disease cases lack a positive family history. 4. Some counties and the eastern part of Hungary shows elevated incidence of human prion disease. CONCLUSIONS: Differences in the geographical distribution may be related to migration and historical relationship with the Slovakian population. Based on the increased incidence of E200K mutation, genetic testing of the PRNP is recommended in all cases with atypical neuropsychiatric disorder or suspicion of prion disease.

Neurons derived from sporadic Alzheimer's disease iPSCs reveal elevated TAU hyperphosphorylation, increased amyloid levels, and GSK3B activation.

BACKGROUND: Alzheimer's disease (AD) is the most common type of dementia, affecting one in eight adults over 65 years of age. The majority of AD cases are sporadic, with unknown etiology, and only 5% of all patients with AD present the familial monogenic form of the disease. In the present study, our aim was to establish an in vitro cell model based on patient-specific human neurons to study the pathomechanism of sporadic AD. METHODS: We compared neurons derived from induced pluripotent stem cell (iPSC) lines of patients with early-onset familial Alzheimer's disease (fAD), all caused by mutations in the PSEN1 gene; patients with late-onset sporadic Alzheimer's disease (sAD); and three control individuals without dementia. The iPSC lines were differentiated toward mature cortical neurons, and AD pathological hallmarks were analyzed by RT-qPCR, enzyme-linked immunosorbent assay, and Western blotting methods. RESULTS: Neurons from patients with fAD and patients with sAD showed increased phosphorylation of TAU protein at all investigated phosphorylation sites. Relative to the control neurons, neurons derived from patients with fAD and patients with sAD exhibited higher levels of extracellular amyloid-ß 1-40 (Aß1-40) and amyloid-ß 1-42 (Aß1-42). However, significantly increased Aß1-42/Aß1-40 ratios, which is one of the pathological markers of fAD, were observed only in samples of patients with fAD. Additionally, we detected increased levels of active glycogen synthase kinase 3 ß, a physiological kinase of TAU, in neurons derived from AD iPSCs, as well as significant upregulation of amyloid precursor protein (APP) synthesis and APP carboxy-terminal fragment cleavage. Moreover, elevated sensitivity to oxidative stress, as induced by amyloid oligomers or peroxide, was detected in both fAD- and sAD-derived neurons. CONCLUSIONS: On the basis of the experiments we performed, we can conclude there is no evident difference except secreted Aß1-40 levels in phenotype between fAD and sAD samples. To our knowledge, this is the first study in which the hyperphosphorylation of TAU protein has been compared in fAD and sAD iPSC-derived neurons. Our findings demonstrate that iPSC technology is suitable to model both fAD and sAD and may provide a platform for developing new treatment strategies for these conditions.

Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells.

Neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs) are frequently induced using 3D culture methodologies however, it is unknown whether spheroid-based (3D) neural induction is actually superior to monolayer (2D) neural induction. Our aim was to compare the efficiency of 2D induction with 3D induction method in their ability to generate NPCs, and subsequently neurons and astrocytes. Neural differentiation was analysed at the protein level qualitatively by immunocytochemistry and quantitatively by flow cytometry for NPC (SOX1, PAX6, NESTIN), neuronal (MAP2, TUBB3), cortical layer (TBR1, CUX1) and glial markers (SOX9, GFAP, AQP4). Electron microscopy demonstrated that both methods resulted in morphologically similar neural rosettes. However, quantification of NPCs derived from 3D neural induction exhibited an increase in the number of PAX6/NESTIN double positive cells and the derived neurons exhibited longer neurites. In contrast, 2D neural induction resulted in more SOX1 positive cells. While 2D monolayer induction resulted in slightly less mature neurons, at an early stage of differentiation, the patch clamp analysis failed to reveal any significant differences between the electrophysiological properties between the two induction methods. In conclusion, 3D neural induction increases the yield of PAX6+/NESTIN+ cells and gives rise to neurons with longer neurites, which might be an advantage for the production of forebrain cortical neurons, highlighting the potential of 3D neural induction, independent of iPSCs' genetic background.

Subcellular distribution of components of the ubiquitin-proteasome system in non-diseased human and rat brain.

Our aim was to investigate and to compare the intracellular distribution of ubiquitin, 20S proteasome, and all six proteasomal regulatory ATPases in non-diseased human and rat brains. Ubiquitin and ATPases S4 and S7 show dominant nuclear immunostaining, whereas subunits S6a, S6b, and S10b show mainly cytoplasmic immunostaining in both species. However, S8 localization is inconsistent, prevailing nuclear in rat and cytoplasmic in human. In rat brain, small clastosome-like nuclear bodies demonstrate strong ubiquitin, 20S, and S6a immunoreactivity both in neurons and glial cells. Prominent nuclear immunolocalization of members of the ubiquitin-proteasome system provides morphological evidence for function of these proteins in transcription regulation and/or DNA repair.

Immunohistochemistry for the prion protein: comparison of different monoclonal antibodies in human prion disease subtypes.

Demonstration of the abnormal form of the prion protein (PrP) in the brain confirms the diagnosis of human prion disease (PrD). Using immunohistochemistry, we have compared ten monoclonal antibodies in PrD subtypes including sporadic and variant Creutzfeldt-Jakob disease (CJD), fatal familial insomnia, Alzheimer's disease (AD), and control brains. CJD subgroups were determined using Western blot analysis for the protease-resistant PrP type in combination with sequencing to determine the genotype at the methionine/valine polymorphism at codon 129 of the prion protein gene. None of the antibodies labeled given subgroups exclusively, but the intensity of immunoreactivity varied among morphologically distinct types of deposit. Fine granular or synaptic PrP deposits stained weakly or not at all with antibodies against the N-terminus of PrP, and were visible in one case only with 12F10 and SAF54. Coarser and plaque type deposits were immunolabeled with all antibodies. The immunostaining patterns appear characteristic for the disease subgroups. Labeling of certain neurons in all cases irrespective of disease, and staining at the periphery and/or throughout the senile plaques of AD patients were also noted. Antibodies such as 6H4 and 12F10 failed to give this type of labeling and are therefore less likely to recognise non-pathological PrP material in immunohistochemistry.

Increased sensitivity to NMDA is involved in alcohol-withdrawal induced cytotoxicity observed in primary cultures of cortical neurones chronically pre-treated with ethanol.

Severe cellular damage and neuronal cell loss were previously observed in cultures of primary cortical neurones after chronic ethanol pre-treatment followed by ethanol-withdrawal. In this study, we investigated the circumstances and the possible cellular changes leading to alcohol-withdrawal induced neuronal cell death. When cultures were pre-treated with ethanol (25-200mM) once for 24 or 72h, the amount of the subsequent 24h alcohol-withdrawal induced cell death-estimated by measuring the release of lactate dehydrogenase (LDH)-was elevated only in cultures pre-treated with 200mM ethanol for 72h. On the contrary, as little as 50mM ethanol produced significant (P<0.01) increase in the withdrawal induced LDH-release in cultures pre-treated repeatedly with ethanol once daily for three consecutive days. When ethanol was re-added to the cultures during the withdrawal period, the LDH-release was dose-dependently reduced to the level of control. In ethanol pre-treated cultures N-methyl-D-aspartate (NMDA) (0.01-1mM) induced excitotoxicity as well as NMDA evoked elevation of cytosolic calcium ion concentration was increased. In contrast, the depolarising agent veratridine (0.01-1mM) produced similar extent of neuronal injury and elevation in cytosolic calcium ion concentration in control as in ethanol pre-treated cultures. According to these observations, repeated ethanol treatment appears to cause more robust adaptive changes in cultured neurones leading to more pronounced withdrawal induced cellular damage than chronic but single treatment does. In addition, the glutamatergic neurotransmission, especially the NMDA receptor system seems to be highly involved in the adaptive changes and in the cytotoxic effect of alcohol-withdrawal.

Serum amyloid P component induces neuronal apoptosis and beta-amyloid immunoreactivity.

Previously we have reported serum amyloid P component (SAP) induced cell death in cerebro-cortical cultures of rat brain. In this paper we studied the types of target cells and the molecular mechanism of SAP-induced cell death. Immuno-electron and light microscopy revealed that SAP penetrates the plasma membrane and translocates selectively into the nuclei of neurons. Neuronal cells with SAP immunoreactivity exhibit the morphological hallmarks of apoptosis in vitro. The apoptotic mechanism of cell death is also supported by the increased Bax/Bcl-2 ratio. In addition to neurotoxic effects, we detected elevated beta-amyloid (Abeta) immunoreactivity following SAP treatment. This study supports the thesis that SAP plays an important role in the pathomechanism of neurodegenerative diseases, including Alzheimer's disease by inducing neuronal apoptosis.

Rapidly progressive dementia with thalamic degeneration and peculiar cortical prion protein immunoreactivity, but absence of proteinase K resistant PrP: a new disease entity?

BACKGROUND: Human prion diseases are a group of rare fatal neurodegenerative conditions with well-developed clinical and neuropathological diagnostic criteria. Recent observations have expanded the spectrum of prion diseases beyond the classically recognized forms. RESULTS: In the present study we report six patients with a novel, apparently sporadic disease characterised by thalamic degeneration and rapidly progressive dementia (duration of illness 2-12 months; age at death: 55-81 years). Light and electron microscopic immunostaining for the prion protein (PrP) revealed a peculiar intraneuritic distribution in neocortical regions. Proteinase K resistant PrP (PrPres) was undetectable by Western blotting in frontal cortex from the three cases with frozen tissue, even after enrichment for PrPres by centrifugation or by phosphotungstic acid precipitation. Conformation-dependent immunoassay analysis using a range of PK digestion conditions (and no PK digestion) produced only very limited evidence of meaningful D-N (denatured/native) values, indicative of the presence of disease-associated PrP (PrPSc) in these cases, when the results were compared with appropriate negative control groups. CONCLUSIONS: Our observation expands the spectrum of conditions associated with rapidly progressive dementia and may have implications for the understanding of the pathogenesis of prion diseases.

Intraneuronal immunoreactivity for the prion protein distinguishes a subset of E200K genetic from sporadic Creutzfeldt-Jakob Disease.

Recently, we reported widespread intraneuronal prion protein (PrP) immunoreactivity in genetic Creutzfeldt-Jakob disease (CJD) associated with the E200K mutation. Here, we evaluated 6 cases ofsporadic CJD MM type 1, 5 MV type 2, and 7 VV type 2 and compared their anatomical appearance with that of 29 E200K genetic CJD (gCJD) cases. We also performed double immunolabeling for ubiquitin, p62, early endosomal marker rab5, and immunogold electronmicroscopy in 3 cases. We identified 4 morphological types of intraneuronal PrP immunoreactivity: one type, defined as multiple globular structures, was significantly associated with a subset of E200K gCJD cases and was distinct from the intraneuronal small dotlike PrP immunoreactivity seen in sporadic CJD. Whereas the latter colocalized with rab5, there were single large (7.5 µm-15 µm) globular inclusion body-like structures detected predominantly but not exclusively in E200K gCJD; these were immunoreactive in part for ubiquitin and p62 and showed focal γ-tubulin immunoreactivity, suggesting aggresome features. Ultrastructural examination using immunogold revealed PrP localization in aggresome-like structures and in autophagic vacuoles. These findings suggest that the permanent production of mutant PrP in the E200K gCJD cases overwhelms the ubiquitin-proteasome system and shifts the balance toward selectivemacroautophagy and/or to ubiquitinated inclusion body and aggresome formation as a cytoprotective effort to sequester the mutant protein.