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

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

High specificity of Hook isoform-specific antibodies and Hook isoform localization in control brain and AD brain.

<p>(a) Affinity purified Hook isoform-specific antibodies were tested on lysates of N2A cells transfected with pEGFP-Hook plasmids. Lysates of N2A cells expressing EGFP-Hook1 (lane 1), EGFP-Hook2 (lane 2), EGFP-Hook3 (lane 3) and EGFP only (lane 4) were separated on 10% SDS-PAGE and transferred to PVDF membrane. The membrane was probed with 0.5 μg/ml anti-Hook1 antiserum (a-Hk1), 0.5 μg/ml anti-Hook2 antiserum (a-Hk2), 0.5 μg/ml anti-Hook3 antiserum (a-Hk3) or 0.25 μg/ml pan-Hook antiserum (a-pan Hk). Pan-Hook antiserum shows the expression of all EGFP-Hook fusion proteins in N2A cells (100–120 kDa), whereas the isoform-specific antisera only label the corresponding isoform. (b) No crossreactivity of affinity-purified Hook antibodies with PHF-tau prepared from AD brain was detected, phospho-tau antibody AT8 (lane1), a-Hook1 (lane 2), a-Hook2 (lane 3), a-Hook3 (lane 4). (c) The Dot blot shows that Hook2 antibody does not crossreact with β-amyloid; 100 ng EGFP-Hook2 N2A cell lysate (spot 1, 3); 100 ng β-amyloid 1–40 (spot 2, 4). (d) Immunohistochemical staining of the hippocampal CA3 region using Hook1, Hook2 and Hook3 antiserum in control brain and in AD brain (Braak stage V). Hook1 and Hook3 antibodies label tau pathology such as neurofibrillary tangles (asterisk) and dystrophic neurites in neuritic plaques (+). Granular neuronal Hook3 immunoreactivity present in control brains (arrowhead) is reduced in AD and NFT-associated Hook3 immunoreactivity is predominant. Amyloid plaque staining (+) is observed using the Hook2 isoform-specific antibody. Scale bar = 20 μm.</p

Hook3 knockdown increases production of endogenous β-amyloid in N2A cells.

<p>(a) N2A cells were transfected with two different siRNAs targeting Hook3 and with non-target siRNA as control (n = 3). Beta-amyloid accumulated for 48h in cell culture media was quantified by a murine amyloid-specific ELISA. Hook3 downregulation by two different siRNAs causes a 35% increase in secreted β-amyloid. * p < 0.05 compared to control group with student’s t-test. Data are means ± SD. (b) Western blot analysis of cell lysates demonstrate that transfection with Hook3 siRNA does not alter the levels of intracellular APP, BACE1 and PS1.</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

Immunohistochemical staining of Hook proteins in brain tissue of 6 month old P301L- tau transgenic mice.

<p>(a) Overview of Hook immunoreactivities in the prefrontal cortex (lateral of the central sulcus). Hook1 antibody shows a weak cytoplasmic immunoreactivity in the cortex, while Hook2 antibody marks distinct punctuate structures. Hook3 antibody clearly labels the soma of neurons. The staining shows a granular pattern. Note the tangle-like structures in the anti-Hook1 stained section (asterisk) (b) Staining of the brainstem with Hook1 and AT100 antibodies; tau-aggregates co-localise with Hook1. (c) Staining of the brainstem with Hook3 and AT100 antibodies. Hook3 antibody marks a neuron containing aggregated tau. (d) Staining of Hook2, GFAP and IBA1 in the brainstem; Hook2 co-localises with GFAP, but not with IBA1. Scale bars = a: 100 μm, b-d: 50 μm.</p

Case details.

<p>Case details.</p