Hook proteins: association with Alzheimer pathology and regulatory role of Hook3 inAmyloid 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 β-amyloid production. We propose that Hook3 plays a role in pathogenic events exacerbating AD

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-Aβ peptides in Alzheimer’s disease—act as a seed for pathogenic protein aggregation

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

AbstractTau 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

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 β-amyloid production. We propose that Hook3 plays a role in pathogenic events exacerbating AD

Hook proteins: association with Alzheimer pathology and regulatory role of Hook3 inAmyloid 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 β-amyloid production. We propose that Hook3 plays a role in pathogenic events exacerbating AD

Hook proteins: association with Alzheimer pathology and regulatory role of Hook3 inAmyloid 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 β-amyloid production. We propose that Hook3 plays a role in pathogenic events exacerbating AD

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 Aβ 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-3β, 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-3β in nanomolar ranges

Hook protein levels in brain tissue of patients with AD.

<p>(a) Western blot analysis of the cytoplasmic fractions of brain homogenates. Grey substance of Brodmann area 22 (temporal cortex) was homogenized in detergent-free buffer and was subsequently ultracentrifuged. Twenty-three μg of protein of each supernatant were loaded on SDS-PAGE, analyzed by Western blot and probed with different antibodies. C: Braak stage 0, I: Braak stage I-III, V: Braak stage V-VI. (b) Densitometric analysis of protein immunoreactivity in brain homogenates of controls (Braak stage 0, n = 8), early stages of AD pathology (Braak stages I-III, n = 6) and late stages (Braak stages V-VI, n = 10). Data are means ± SEM. Statistical analysis was carried out with Kruskal-Wallis test and subsequently Mann-Whitney U test. * p < 0.05, *** p < 0.001 compared to control group with Mann-Whitney U test. OD: optical density, WB: Western blot. (c) Quantification of total Hook1 and total Hook3 in whole homogenates by ELISA. Whole homogenates comprising the soluble and the particulate fraction were prepared from tissue of Brodmann area 22 in detergent-free buffer. Hook proteins were captured from whole homogenates with isoform-specific Hook antibodies and detected with biotinylated pan-Hook antibody. Data were corrected by the negative control of the antibody background and groups were statistically tested with Kruskal-Wallis test and Mann-Whitney U test. Data are means ± SEM. (d) Association of Hook1 and Hook3 with PHF-tau analysed by a Sandwich ELISA. Hook proteins were captured from whole homogenates with anti-Hook1 or anti-Hook3 antibody. Detection with AT100 revealed an association of Hook1 and Hook3 with PHF-tau in four brain homogenates from patients with AD. (e) Hook3 binding to recombinant tau is increased by tau aggregation and not by tau phosphorylation. Recombinant Hook3 was incubated with 2N4R-tau either in its unmodified, aggregated (aggr.) or phosphorylated form (p-tau) or with both modifications and applied to a monoclonal pan-tau capturing antibody. Hook3 protein associated with captured tau protein was quantified using a polyclonal Hook3 antibody in this sandwich-ELISA (left panel). Hook3 immunoreactivity was corrected for background reactivity and equal binding of the different modified tau species was verified by detection with the polyclonal tau antibody (right panel).</p

Case details.

<p>Case details.</p

Localization of Hook1, Hook2 and Hook3 in relation to pathological markers in AD brain.

<p>(a) Hook1 detection in the hippocampus reveals strong labeling of neurofibrillary tangles (asterisk) and dystrophic neurites of neuritic plaques (+) co-labeled by AT8. Dystrophic neurites are detected more clearly by anti-Hook1 compared to AT8. (b) Hook2 protein is enriched in cellular components within amyloid plaques detected by anti-amyloid antibody 6F3D (+), but not overlapping with β-amyloid localization. Hook2 is not associated with tau aggregates. (c) Hook3 is expressed in neurons and labeling is increased in tangle-bearing neurons (asterisk) and dystrophic neurites (+) detected with AT8. Scale bar = 20 μm.</p