Senile plaque calcification of the lamina circumvoluta medullaris in Alzheimer's disease.

Vascular calcification is a common phenomenon in the elderly, predominantly appearing in the basal ganglia and in the lamina circumvoluta medullaris of the hippocampus. Calcifications are not an inherent feature of Alzheimer's disease. On the other hand, a rare presenile type of dementia with symmetrical Fahr-type calcifications and numerous neurofibrillary tangles without senile plaques has been described by Kosaka in 1994 and was termed "diffuse neurofibrillary tangles with calcification" (DNTC). We here report a case of Alzheimer's disease with calcifications both in the basal ganglia and in the lamina circumvoluta medullaris of the hippocampus, differing from DNTC by the presence of senile plaques. The calcifications in the hippocampus were not only vascular in nature but also covered amyloid-ß- and phosphorylated tau-positive plaque-like structures that were linearly arranged along the dentate fascia in the CA1 sector, an unusual finding of pathogenetic interest.

A glutaminyl cyclase-catalyzed α-synuclein modification identified in human synucleinopathies.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that is neuropathologically characterized by degeneration of dopaminergic neurons of the substantia nigra (SN) and formation of Lewy bodies and Lewy neurites composed of aggregated α-synuclein. Proteolysis of α-synuclein by matrix metalloproteinases was shown to facilitate its aggregation and to affect cell viability. One of the proteolysed fragments, Gln79-α-synuclein, possesses a glutamine residue at its N-terminus. We argue that glutaminyl cyclase (QC) may catalyze the pyroglutamate (pGlu)79-α-synuclein formation and, thereby, contribute to enhanced aggregation and compromised degradation of α-synuclein in human synucleinopathies. Here, the kinetic characteristics of Gln79-α-synuclein conversion into the pGlu-form by QC are shown using enzymatic assays and mass spectrometry. Thioflavin T assays and electron microscopy demonstrated a decreased potential of pGlu79-α-synuclein to form fibrils. However, size exclusion chromatography and cell viability assays revealed an increased propensity of pGlu79-α-synuclein to form oligomeric aggregates with high neurotoxicity. In brains of wild-type mice, QC and α-synuclein were co-expressed by dopaminergic SN neurons. Using a specific antibody against the pGlu-modified neo-epitope of α-synuclein, pGlu79-α-synuclein aggregates were detected in association with QC in brains of two transgenic mouse lines with human α-synuclein overexpression. In human brain samples of PD and dementia with Lewy body subjects, pGlu79-α-synuclein was shown to be present in SN neurons, in a number of Lewy bodies and in dystrophic neurites. Importantly, there was a spatial co-occurrence of pGlu79-α-synuclein with the enzyme QC in the human SN complex and a defined association of QC with neuropathological structures. We conclude that QC catalyzes the formation of oligomer-prone pGlu79-α-synuclein in human synucleinopathies, which may-in analogy to pGlu-Aß peptides in Alzheimer's disease-act as a seed for pathogenic protein aggregation.

Novel Protein Kinase Inhibitors Related to Tau Pathology Modulate Tau Protein-Self Interaction Using a Luciferase Complementation Assay.

The current number of drugs available for the treatment of Alzheimer's disease (AD) is strongly limited and their benefit for therapy is given only in the early state of the disease. An effective therapy should affect those processes which mainly contribute to the neuronal decay. There have been many approaches for a reduction of toxic Aß peptides which mostly failed to halt cognitive deterioration in patients. The formation of neurofibrillary tangles (NFT) and its precursor tau oligomers have been suggested as main cause of neuronal degeneration because of a direct correlation of their density to the degree of dementia. Reducing of tau aggregation may be a viable approach for the treatment of AD. NFT consist of hyperphosphorylated tau protein and tau hyperphosphorylation reduces microtubule binding. Several protein kinases are discussed to be involved in tau hyperphosphorylation. We developed novel inhibitors of three protein kinases (gsk-3ß, cdk5, and cdk1) and discussed their activity in relation to tau phosphorylation and on tau⁻tau interaction as a nucleation stage of a tau aggregation in cells. Strongest effects were observed for those inhibitors with effects on all the three kinases with emphasis on gsk-3ß in nanomolar ranges.

Tau phosphorylation-associated spine regression does not impair hippocampal-dependent memory in hibernating golden hamsters.

The microtubule-associated protein tau, in its hyperphosphorylated form, is the major component of paired helical filaments and other aggregates in neurodegenerative disorders commonly referred to as "tauopathies". Recent evidence, however, indicates that mislocalization of hyperphosphorylated tau to subsynaptic sites leads to synaptic impairment and cognitive decline even long before formation of tau aggregates and neurodegeneration occur. A similar, but reversible hyperphosphorylation of tau occurs under physiologically controlled conditions during hibernation. Here, we study the hibernating Golden hamster (Syrian hamster, Mesocricetus auratus). A transient spine reduction was observed in the hippocampus, especially on apical dendrites of hippocampal CA3 pyramidal cells, but not on their basal dendrites. This distribution of structural synaptic regression was correlated to the distribution of phosphorylated tau, which was highly abundant in apical dendrites but hardly detectable in basal dendrites. Surprisingly, hippocampal memory assessed by a labyrinth maze was not affected by hibernation. The present study suggests a role for soluble hyperphosphorylated tau in the process of reversible synaptic regression, which does not lead to memory impairment during hibernation. We hypothesize that tau phosphorylation associated spine regression might mainly affect unstable/dynamic spines while sparing established/stable spines.

Hook proteins: association with Alzheimer pathology and regulatory role of hook3 in amyloid beta generation.

Defects in intracellular transport are implicated in the pathogenesis of Alzheimer's disease (AD). Hook proteins are a family of cytoplasmic linker proteins that participate in endosomal transport. In this study we show that Hook1 and Hook3 are expressed in neurons while Hook2 is predominantly expressed in astrocytes. Furthermore, Hook proteins are associated with pathological hallmarks in AD; Hook1 and Hook3 are localized to tau aggregates and Hook2 to glial components within amyloid plaques. Additionally, the expression of Hook3 is reduced in AD. Modelling of Hook3 deficiency in cultured cells leads to slowing of endosomal transport and increases ß-amyloid production. We propose that Hook3 plays a role in pathogenic events exacerbating AD.

Axotrophin/MARCH7 acts as an E3 ubiquitin ligase and ubiquitinates tau protein in vitro impairing microtubule binding.

Tau is the major microtubule-associated protein in neurons involved in microtubule stabilization in the axonal compartment. Changes in tau gene expression, alternative splicing and posttranslational modification regulate tau function and in tauopathies can result in tau mislocalization and dysfunction, causing tau aggregation and cell death. To uncover proteins involved in the development of tauopathies, a yeast two-hybrid system was used to screen for tau-interacting proteins. We show that axotrophin/MARCH7, a RING-variant domain containing protein with similarity to E3 ubiquitin ligases interacts with tau. We defined the tau binding domain to amino acids 552-682 of axotrophin comprising the RING-variant domain. Co-immunoprecipitation and co-localization confirmed the specificity of the interaction. Intracellular localization of axotrophin is determined by an N-terminal nuclear targeting signal and a C-terminal nuclear export signal. In AD brain nuclear localization is lost and axotrophin is rather associated with neurofibrillary tangles. We find here that tau becomes mono-ubiquitinated by recombinant tau-interacting RING-variant domain, which diminishes its microtubule-binding. In vitro ubiquitination of four-repeat tau results in incorporation of up to four ubiquitin molecules compared to two molecules in three-repeat tau. In summary, we present a novel tau modification occurring preferentially on 4-repeat tau protein which modifies microtubule-binding and may impact on the pathogenesis of tauopathies.

Pin1 promotes degradation of Smad proteins and their interaction with phosphorylated tau in Alzheimer's disease.

AIMS: Neurodegeneration in Alzheimer's disease (AD) is characterized by pathological protein aggregates and inadequate activation of cell cycle regulating proteins. Recently, Smad proteins were identified to control the expression of AD relevant proteins such as APP, CDK4 and CDK inhibitors, both critical regulators of cell cycle activation. This might indicate a central role for Smads in AD pathology where they show a substantial deficiency and disturbed subcellular distribution in neurones. Still, the mechanisms driving relocation and decrease of neuronal Smad in AD are not well understood. However, Pin1, a peptidyl-prolyl-cis/trans-isomerase, which allows isomerization of tau protein, was recently identified also controlling the fate of Smads. Here we analyse a possible role of Pin1 for Smad disturbances in AD. METHODS: Multiple immunofluorescence labelling and confocal laser-scanning microscopy were performed to examine the localization of Smad and Pin1 in human control and AD hippocampi. Ectopic Pin1 expression in neuronal cell cultures combined with Western blot analysis and immunoprecipitation allowed studying Smad level and subcellular distribution. Luciferase reporter assays, electromobility shift, RNAi-technique and qRT-PCR revealed a potential transcriptional impact of Smad on Pin1 promoter. RESULTS: We report on a colocalization of phosphorylated Smad in AD with Pin1. Pin1 does not only affect Smad phosphorylation and stability but also regulates subcellular localization of Smad2 and supports its binding to phosphorylated tau protein. Smads, in turn, exert a negative feed-back regulation on Pin1. CONCLUSION: Our data suggest both Smad proteins and Pin1 to be elements of a vicious circle with potential pathogenetic significance in AD.

Tau oligomers impair artificial membrane integrity and cellular viability.

The microtubule-associated protein Tau is mainly expressed in neurons, where it binds and stabilizes microtubules. In Alzheimer disease and other tauopathies, Tau protein has a reduced affinity toward microtubules. As a consequence, Tau protein detaches from microtubules and eventually aggregates into ß-sheet-containing filaments. The fibrillization of monomeric Tau to filaments is a multistep process that involves the formation of various aggregates, including spherical and protofibrillar oligomers. Previous concepts, primarily developed for Aß and α-synuclein, propose these oligomeric intermediates as the primary cytotoxic species mediating their deleterious effects through membrane permeabilization. In the present study, we thus analyzed whether this concept can also be applied to Tau protein. To this end, viability and membrane integrity were assessed on SH-SY5Y neuroblastoma cells and artificial phospholipid vesicles, treated with Tau monomers, Tau aggregation intermediates, or Tau fibrils. Our findings suggest that oligomeric Tau aggregation intermediates are the most toxic compounds of Tau fibrillogenesis, which effectively decrease cell viability and increase phospholipid vesicle leakage. Our data integrate Tau protein into the class of amyloidogenic proteins and enforce the hypothesis of a common toxicity-mediating mechanism for amyloidogenic proteins.

Transcriptional control of cell cycle-dependent kinase 4 by Smad proteins--implications for Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by deregulation of neuronal cell cycle and differentiation control eventually resulting in cell death. During brain development, neuronal differentiation is regulated by Smad proteins, which are elements of the canonical transforming growth factor ß (TGF-ß) signaling pathway, linking receptor activation to gene expression. In the normal adult brain, Smad proteins are constitutively phosphorylated and predominantly localized in neuronal nuclei. Under neurodegenerative conditions such as AD, the subcellular localization of their phosphorylated forms is heavily disturbed, raising the question of whether a nuclear Smad deficiency in neurons might contribute to a loss of neuronal differentiation control and subsequent cell cycle re-entry. Here, we show by luciferase reporter assays, electromobility shift, and RNA interference (RNAi) technique a direct binding of Smad proteins to the CDK4 promoter inducing transcriptional inhibition of cell cycle-dependent kinase 4 (Cdk4). Mimicking the neuronal deficiency of Smad proteins observed in AD in cell culture by RNAi results in elevation of Cdk4 and retardation of neurite outgrowth. The results identify Smad proteins as direct transcriptional regulators of Cdk4 and add further evidence to a Smad-dependent deregulation of Cdk4 in AD, giving rise to neuronal dedifferentiation and cell death.