Jens Stieler

AβPP induces cdk5-dependent tau hyperphosphorylation in transgenic mice Tg2576

Previous studies of Abeta-induced neuronal damage of hippocampal cells in culture have provided strong evidence that deregulation of the Cdk5/p35 kinase system is involved in the neurodegeneration pathway. Cdk5 inhibitors and antisense probes neuroprotected hippocampal cells against the neurotoxic action of Abeta. To further investigate the mechanisms underlying the participation of Cdk5 in neuronal degeneration, the transgenic mouse containing the Swedish mutations, Tg2576, was used as an animal model. Immunocytochemical studies using anti-Abeta(1-17) antibody evidenced the presence of labeled small-clustered core plaques in the hippocampus and cortex of 18-month-old transgenic mice brains. The loss of granular cells without a compressed appearance was detected in the vicinity of the cores in the dentate gyrus of the hippocampus. Immunostaining of Tg2576 brain sections with antibodies AT8, PHF1 and GFAP labeled punctuate dystrophic neurites in and around the amyloid core. Reactive astrogliosis around the plaques in the hippocampus was also observed. Studies at the molecular level showed differences in the expression of the truncated Cdk5 activator p25 in the transgenic animal, as compared with wild type controls. However no differences in Cdk5 levels were detected, thus corroborating previous cellular findings. Interestingly, hyperphosphorylated tau epitopes were substantially increased as assessed with the AT8 and PHF1 antibodies, in agreement with the observation of a p25 increase in the transgenic animal. These observations strongly suggest that the increased exposure of Alzheimers type tau phosphoepitopes in the transgenic mice correlated with deregulation of Cdk5 leading to an increase in p25 levels. These studies also provide further evidence on the links between extraneuronal amyloid deposition and tau pathology.

Pseudophosphorylation of tau protein alters its ability for self-aggregation

Filamentous tau protein deposits are a pathological hallmark of a group of neurodegenerative disorders (tauopathies). Tau protein in these aggregates is highly phosphorylated at different phosphorylation sites. Although tau filaments can be formed by heparin‐induced aggregation of unphosphorylated recombinant tau, it is not known how tau phosphorylation modulates aggregation behaviour. Analysis of the effect of tau phosphorylation at defined single or multiple sites is hampered by the low specificity of protein kinases and the highly dynamic turnover of phosphorylation in vivo. To overcome this problem we employed site‐directed mutagenesis to convert serine and threonine to aspartic acid or glutamic acid, which introduce a negative charge and conformational change that mimic phosphorylation. We tested 14 different mutated tau proteins for their propensity for self‐aggregation and formation of tau filaments. Tau aggregation was monitored with thioflavin S fluorescence in the presence of different inducers such as heparin, Al3+, Fe2+ and Fe3+. We found that mutations in the N‐terminal portion up to amino acid 208 mainly suppress tau aggregation, whereas mutations in the C‐terminal region mainly lead to an enhanced aggregation. Mutations in the middle portion of tau showed a mixed picture of suppression and enhancement of aggregation. A single amino acid change Ser422Glu has aggregation‐favouring properties with all four inducers.

Tau and tauopathies.

Most neurodegenerative diseases are characterized by intracellular aggregates of insoluble proteins. As for the majority of these disorders, aetiology and pathogenesis are only poorly understood; current nosological concepts are largely based on these molecular signatures of protein aggregates which also provide valuable tools for neuropathological differential diagnosis. The microtubule associated protein tau is one of these proteins that form intracellular fibrillary deposits in neurons and glial cells of a large variety of disorders today collectively referred to as tauopathies. While dysfunction of tau has unequivocally been shown to be able to cause neurodegeneration, the precise mechanisms of how tau is involved in neurodegenerative disorders is still poorly understood. After research has focused for several decades on the axonal function of tau and on the fibrillar tau aggregation, more recent cell biological studies have opened up new insights into the role of tau at the synapse and in the nucleus. According to currently emerging cell biological concepts, tau might play a role in the regulation of neuronal plasticity in a wide array of neuronal networks. In addition, it might be involved in regulating genome stability. The most intriguing question relevant both to physiological and pathophysiological function of tau is the biological meaning of the large heterogeneity of isoforms of tau which apparently is a rather promiscuous molecule. The present review is divided into two parts. First, we give an overview on the molecular biology and cell biology of tau and its physiological functions. The second part deals with the pathophysiology of tau and description of tauopathies which comprise more than 20 disorders including Alzheimers disease, progressive supranuclear palsy, cortico basal degeneration, Picks disease and others.

Hibernation model of tau phosphorylation in hamsters: selective vulnerability of cholinergic basal forebrain neurons – implications for Alzheimer's disease

Neurofibrillar tangles made up of ‘paired helical filaments’ (PHFs) consisting of hyperphosphorylated microtubule‐associated protein tau are major hallmarks of Alzheimers disease (AD). Tangle formation selectively affects certain neuronal types and systematically progresses throughout numerous brain areas, which reflects a hierarchy of neuronal vulnerability and provides the basis for the neuropathological staging of disease severity. Mechanisms underlying this selective neuronal vulnerability are unknown. We showed previously that reversible PHF‐like phosphorylation of tau occurs during obligate hibernation. Here we extend these findings to facultative hibernators such as Syrian hamsters (Mesocricetus auratus) forced into hibernation. In this model, we showed in the basal forebrain projection system that cholinergic neurons are selectively affected by PHF‐like phosphorylated tau, while γ‐aminobutyric acid (GABA)ergic neurons are largely spared, which shows strong parallels to the situation in AD. Formation of PHF‐tau in these neurons apparently does not affect their function as pacemaker for terminating hibernation. We conclude that although formation of PHF‐like phosphorylated tau in the mammalian brain follows a certain hierarchy, affecting some neurons more frequently than others, it is not necessarily associated with impaired neuronal function and viability. This indicates a more general link between PHF‐like phosphorylation of tau and the adaptation of neurons under conditions of a ‘vita minima’.

The Physiological Link between Metabolic Rate Depression and Tau Phosphorylation in Mammalian Hibernation

Abnormal phosphorylation and aggregation of tau protein are hallmarks of a variety of neurological disorders, including Alzheimers disease (AD). Increased tau phosphorylation is assumed to represent an early event in pathogenesis and a pivotal aspect for aggregation and formation of neurofibrillary tangles. However, the regulation of tau phosphorylation in vivo and the causes for its increased stage of phosphorylation in AD are still not well understood, a fact that is primarily based on the lack of adequate animal models. Recently we described the reversible formation of highly phosphorylated tau protein in hibernating European ground squirrels. Hence, mammalian hibernation represents a model system very well suited to study molecular mechanisms of both tau phosphorylation and dephosphorylation under in vivo physiological conditions. Here, we analysed the extent and kinetics of hibernation-state dependent tau phosphorylation in various brain regions of three species of hibernating mammals: arctic ground squirrels, Syrian hamsters and black bears. Overall, tau protein was highly phosphorylated in torpor states and phosphorylation levels decreased after arousal in all species. Differences between brain regions, hibernation-states and phosphosites were observed with respect to degree and kinetics of tau phosphorylation. Furthermore, we tested the phosphate net turnover of tau protein to analyse potential alterations in kinase and/or phosphatase activities during hibernation. Our results demonstrate that the hibernation-state dependent phosphorylation of tau protein is specifically regulated but involves, in addition, passive, temperature driven regulatory mechanisms. By determining the activity-state profile for key enzymes of tau phosphorylation we could identify kinases potentially involved in the differentially regulated, reversible tau phosphorylation that occurs during hibernation. We show that in black bears hibernation is associated with conformational changes of highly phosphorylated tau protein that are typically related to neuropathological alterations. The particular hibernation characteristics of black bears with a continuous torpor period and an only slightly decreased body temperature, therefore, potentially reflects the limitations of this adaptive reaction pattern and, thus, might indicate a transitional state of a physiological process.

The unfolded protein response affects neuronal cell cycle protein expression: implications for Alzheimer's disease pathogenesis

Alzheimers disease (AD) is characterized by the accumulation and aggregation of misfolded proteins. The presence of misfolded proteins in the endoplasmic reticulum (ER) triggers a cellular stress response called the unfolded protein response (UPR). Previously, we have shown that the UPR is activated in AD neurons. In actively dividing cells, activation of the UPR is accompanied by decreased cell cycle protein expression and an arrest in the G1 phase of the cell cycle. Aberrant expression of cell cycle proteins has been observed in post mitotic neurons in AD and is suggested to be involved in neurodegeneration. In this study we show that the protein levels of BiP/GRP78, an ER-stress marker, is increased in Braak stages B and C for amyloid deposits. This is in contrast to the levels of cell cycle markers cyclin D1, cyclin E and phosphorylated retinoblastoma protein (ppRb) which are decreased in Braak stage C compared to Braak stage A for amyloid deposits. In addition, we report a negative correlation between neuronal expression of ppRb and expression levels of BiP/GRP78 in control and AD cases. Activation of the UPR in neuronal cells induces changes in cell cycle protein expression similar to these observed in AD brain. ER stress inducers tunicamycin and thapsigargin down-regulate cell cycle proteins ppRb and cyclin D1 in differentiated neuroblastoma cells. In contrast, protein levels of p27, a cyclin dependent kinase inhibitor, are increased after induction of ER-stress using tunicamycin. These data suggest that activation of the UPR affects cell cycle protein expression in neurons during neurodegeneration in AD.

Impairment of mitogenic activation of peripheral blood lymphocytes in Alzheimer's disease.

Cell-cycle dysregulation might be critically involved in the process of neurodegeneration in Alzheimers disease (AD). We now provide evidence for a dysfunction of the cell division cycle as a more general cellular phenomenon of the disease. Peripheral blood lymphocytes, stimulated with mitogenic compounds, were less able to express CD69, an early proliferation marker, in AD patients than in age-matched controls. Expression levels of CD69 of both T-cells and B-cells correlated inversely with the Mini-mental Scale. The results suggest that a systemic failure of cellular proliferation control might be of critical importance for the pathomechanism of AD.

Alix regulates egress of hepatitis B virus naked capsid particles in an ESCRT-independent manner

Hepatitis B virus (HBV) is an enveloped DNA virus that exploits the endosomal sorting complexes required for transport (ESCRT) pathway for budding. In addition to infectious particles, HBV‐replicating cells release non‐enveloped (nucleo)capsids, but their functional implication and pathways of release are unclear. Here, we focused on the molecular mechanisms and found that the sole expression of the HBV core protein is sufficient for capsid release. Unexpectedly, released capsids are devoid of a detectable membrane bilayer, implicating a non‐vesicular exocytosis process. Unlike virions, naked capsid budding does not require the ESCRT machinery. Rather, we identified Alix, a multifunctional protein with key roles in membrane biology, as a regulator of capsid budding. Ectopic overexpression of Alix enhanced capsid egress, while its depletion inhibited capsid release. Notably, the loss of Alix did not impair HBV production, furthermore indicating that virions and capsids use diverse export routes. By mapping of Alix domains responsible for its capsid release‐mediating activity, its Bro1 domain was found to be required and sufficient. Alix binds to core via its Bro1 domain and retained its activity even if its ESCRT‐III binding site is disrupted. Together, the boomerang‐shaped Bro1 domain of Alix appears to escort capsids without ESCRT.

Hippocampal synaptophysin immunoreactivity is reduced during natural hypothermia in ground squirrels

Natural hypothermia during hibernation results in physiological and behavioral deficits. These changes may be traced at the level of hippocampal signal transduction. We investigated synaptophysin immunoreactivity (SYN-ir) in the hippocampus after short and long periods of hypothermia and short and long periods of euthermy in hibernating ground squirrels. SYN-ir in the stratum lucidum of the hippocampus was transiently reduced during natural hypothermia. Natural hypothermia thus reduces synaptic efficacy. This may play a role in the reduced neuronal connectivity of CA3 pyramidal cell dendrites observed in hibernating ground squirrels.

Reelin-immunoreactive Cajal-Retzius cells: the entorhinal cortex in normal aging and Alzheimer's disease

Alzheimers disease (AD) is a disorder of brain self organization associated with morphodysregulation at the synaptic level. Disturbances follow a hierarchical spatio-temporal pattern throughout the cortex and involve the re-activation of developmental molecular programs. The large glycoprotein reelin, synthesized by Cajal-Retzius (CR) cells, is an important component of a signaling pathway involved in embryonic development and modulation of synaptic circuitry, but is also implicated in the pathogenetic cascade in AD. Although the majority of CR cells sequentially disappears from the postnatal cortical layer I, a few of them persist in the normal adult brain. They continue to produce reelin, express a variety of other proteins, and are characterized by a typical morphology. Recently, CR cells have been reported to be altered in number and morphology in a variety of neurological and psychiatric diseases linked to maldevelopment. In the present study we show that reelin-positive CR cells persist in the layer I of the entorhinal cortex in normal senescent brains and are also preserved in AD. The majority of CR cells in AD is morphologically and cytochemically—as revealed by double labeling with calcium-binding proteins—indistinguishable from normal cases, suggesting that they are not dramatically altered in the entorhinal cortex of AD patients. Nevertheless, CR cells seem to be partially affected by the formation of paired helical filaments, indicating subtle changes that are suggested to be a result rather than a cause of the pathogenetic cascade of AD.