Lajos László

Natively unfolded tubulin polymerization promoting protein TPPP/p25 is a common marker of alpha-synucleinopathies

The novel basic, heat-stable tubulin polymerization promoting protein TPPP/p25 is associated with microtubules in vitro and can induce the formation of aberrant microtubule assemblies. We show by 1H-NMR spectroscopy that TPPP/p25 is natively unfolded. Antisera against peptide 186GKGKAGRVDLVDESG200NH2 (186-200) are highly specific to TPPP/p25. Immunohistochemistry and confocal microscopy demonstrates that TPPP/p25 is enriched in filamentous alpha-synuclein bearing Lewy bodies of Parkinsons (PD) and diffuse Lewy body disease (DLBD), as well as glial inclusions of multiple system atrophy (MSA). There is a correlation between TPPP/p25 and alpha-synuclein immunoreactivity in Western blot. In contrast, TPPP/p25 is not associated with abnormally phosphorylated tau in various inclusions of Picks disease (PiD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). However, electron microscopy confirms clusters of TPPP/p25 immunoreactivity along filaments of unstructured but not compact neurofibrillary tangles in Alzheimers disease (AD). TPPP/p25 seems to be a novel marker of alpha-synucleinopathies.

Ubiquitinated protein conjugates are specifically enriched in the lysosomal system of fibroblasts

Ubiquitin‐protein conjugates are found by imniunogold electron microscopy to be enriched (12‐fold) in the lysosomal compartment of 3T3‐L1 fibroblasts. Treatment of fibroblasts with the cysteine protease inhibitor E‐64 leads to an expansion of the lysosomal compartment and as a result an increase in the cellular content of ubiquitin‐protein conjugates. There is no change in the specific enrichment of ubiquitin‐protein conjugates in the lysosomal compartment following E‐64 treatment. The results suggest that some ubiquitin‐protein conjugates may normally be degraded lysosomally following sequestration by microautophagy and imply that protein ubiquitination may be one of the signals for protein uptake into lysosomes.

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-β 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 PrPres detected by immunoblotting. Unique and prominent intraneuronal PrP deposition involving brainstem nuclei was also noted. Systematic examination of protein depositions revealed parenchymal amyloid-β in 53.8%, amyloid angiopathy (Aβ) 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.

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

Dementia with Lewy bodies (DLB), Parkinsons 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 β-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 β-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.

Ultrastructural localization of Hsp-72 examined with a new polyclonal antibody raised against the truncated variable domain of the heat shock protein

In spite of the intensive search for the determination of the continuously widening physiological and pathological roles of different stress proteins, their ultrastructural localization at the electron microscopic (EM) level has hardly been examined. As it becomes increasingly evident that the function and physiological effectiveness of stress proteins are highly dependent on their spatial location and their associations with diverse regulator proteins, the demand for morphological studies which can identify their detailed distribution within the cells is evident. The reason for the practical lack of studies carried out at the EM level, lies in the shortage of reagents with suitable specificity and avidity necessary for this type of examination. To create such a reagent, a polyclonal antibody was raised using a recombinant truncated form of the inducible Hsp-72 protein. The antibody was extensively characterized, using different immunochemical methods to determine and verify its specificity, and then it was tried in ultrastructural examinations. Using the new antibody, it was possible to analyze the intracellular distribution of Hsp-72 with the immunogold technique. The localization of Hsp-72 was demonstrated directly at the ultrastructural level in the cytoplasm (especially at the cisterns of the RER), in the nucleus (mainly around the heterochromatic regions) and at both sides of the nuclear envelope close to the membrane pores. Apart from these localizations, Hsp-72 was found in several membrane bordered intracellular structures, which mainly belong to the endosomal-lysosomal system. We provide the first morphological verification of the appearance of Hsp-72 on the surface of the cells. Also novel is the indication, that the stress protein may recycle from the cell surface using a common route which includes coated pits and the endosomal system.

Immunogold localisation of ubiquitin‐protein conjugates in primary (azurophilic) granules of polymorphonuclear neutrophils

Ubiquitin—protein conjugates are found in the primary (azurophilic) lysome‐related granules but not in the secondary (specific) granules in mature polymorphonuclear neutrophils prepared from bone marrow. This is the first reported demonstration of ubiquitin—protein conjugates in lysosome‐related membrane‐bound vesicles in granulocytes and complements our previous findings of ubiquitinated proteins in lysosomes of fibroblasts. The significance of the selective presence of conjugates in only one of the two main types of neutrophil granules remains to be elucidated but may relate to the presence of the complement of acid hydrolases, including proteases, in the azurophilic granules compared to the specific granules. Ubiquitin—protein conjugates may enter the primary granules during neutrophil maturation by an autophagic process or by a heterophagic process during the fusion of phagosomes with primary granules. Alternatively protein ubiquitination may be involved in granule biogenesis.

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.

Effect of amino acids and cycloheximide on changes caused by vinblastine, leupeptin and methylamine in the autophagic/lysosomal system of mouse hepatocytes in vivo

The number of autophagic vacuoles in hepatocytes of 24 h fasted mice in vivo increased manyfold following the administration of vinblastine, leupeptin and methylamine. The effect of each chemical is characterized by the predominance of a certain kind of vacuole. Vinblastine treatment is accompanied by a large proportion of vacuoles containing morphologically unaltered organelles, leupeptin causes preferential accumulation of dense and complex vacuoles, methylamine administration produces mostly large, electron-lucent, swollen vacuoles. The amounts of segregated and accumulated cytoplasmic material, expressed as percentage cytoplasm per hour, were 0.84%, 2.08% and 0.74% following vinblastine, leupeptin and methylamine treatment respectively. The actual rate of segregation was probably higher than this. Inhibition of degradation of the sequestered cytoplasmic material is proposed to be a main factor in the increase in the size of the autophagic/lysosomal compartment. Treatment with cycloheximide or exogenously added mixture of amino acids cut down the size of the autophagic/lysosomal system in control cells and strongly inhibited the accumulation caused by vinblastine, leupeptin and methylamine.

Cytotoxic effect of alcohol-withdrawal on primary cultures of cortical neurones

Physical dependence on alcohol was observed previously at the cellular level in cultured IM-9 human lymphoblast cells. To answer the question whether physical dependence can also develop in neurones and to investigate the neuronal processes involved in the development of alcohol dependence and withdrawal symptoms, cultures of cortical neurones were adapted to alcohol. Morphological characteristics of neurones were not altered during the chronic (3-day) repeated (once per day) ethanol (50-100 mM) treatment, whereas obvious signs of neuronal damage were seen after the following 24 h of alcohol-withdrawal. The extent of the damage, quantitated by measuring the release of lactate dehydrogenase (LDH) into the culture media, was dependent on the concentration of ethanol in the medium during adaptation. LDH-release induced by alcohol-withdrawal was significantly reduced by re-addition of ethanol, as well as by administration of non-competitive (MK-801) or NR2B selective (threo-ifenprodil) N-methyl-D-aspartate (NMDA) receptor antagonists. The sigma ligand haloperidol and the L-type voltage sensitive calcium channel blocker nimodipine were also effective, whereas the effect of the gamma-aminobutyric acid type A (GABA(A)) receptor agonist muscimol was not significant. Furthermore, chronic ethanol treatment potentiated the NMDA induced neurotoxicity and the ability of acute alcohol to inhibit LDH-release in response to NMDA. According to these results, (i) the phenomenon of alcohol-dependence can be observed at the level of neurones and (ii) NMDA receptors seem to play a central role in the development of ethanol dependence and in neurotoxicity induced by alcohol-withdrawal.

Morphometric evaluation of the turnover of autophagic vacuoles after treatment with triton X-100 and vinblastine in murine pancreatic acinar and seminal vesicle epithelial cells

SummaryLarge numbers of autophagic vacuoles were found in murine pancreatic acinar and seminal vesicle epithelial cells following the administration of Triton X-100 or vinblastine for 4 h. The autophagic vacuoles disappeared rapidly from the cells after the administration of cycloheximide to animals pretreated with Triton X-100. The decay in seminal vesicle cells appeared to follow firstorder kinetics with an estimated t1/2 of 8.7 min. The regression in pancreatic cells was equally rapid and less than half the initial volume of autophagic vacuoles was found at the 12th min after cycloheximide injection. This time, the decay curve appeared to be linear rather than exponential. Our data, together with the work of others, support the view that the average half-life of autophagic vacuoles is a fairly constant parameter kept within the range of 6–9 min in various types of mouse and rat cell when the late steps of autophagocytosis (i.e. the fusion of autophagosomes and lysosomes and the degradation within lysosomes) are not affected.The regression of autophagic vacuoles was slow in mice pretreated with vinblastine t1/2 of about 27–30 min) suggesting that this drug slows down the turnover of autophagic vacuoles.Morphometric evaluation of the regression of the autophagic vacuole compartment after cycloheximide treatment can be used as a tool to distinguish between treatments which elevate the amount of autophagic vacuoles within the cells by increasing the rate of sequestration from those which expand the autophagic vacuole compartment by causing accumulation of autophagic vacuoles as a result of blockade of the late steps of the autophagic process.