Uwe Konietzko

Antibodies against β-Amyloid Slow Cognitive Decline in Alzheimer's Disease

To test whether antibodies against beta-amyloid are effective in slowing progression of Alzheimers disease, we assessed cognitive functions in 30 patients who received a prime and a booster immunization of aggregated Abeta(42) over a 1 year period in a placebo-controlled, randomized trial. Twenty patients generated antibodies against beta-amyloid, as determined by tissue amyloid plaque immunoreactivity assay. Patients who generated such antibodies showed significantly slower rates of decline of cognitive functions and activities of daily living, as indicated by the Mini Mental State Examination, the Disability Assessment for Dementia, and the Visual Paired Associates Test of delayed recall from the Wechsler Memory Scale, as compared to patients without such antibodies. These beneficial clinical effects were also present in two of three patients who had experienced transient episodes of immunization-related aseptic meningoencephalitis. Our results establish that antibodies against beta-amyloid plaques can slow cognitive decline in patients with Alzheimers disease.

Generation of antibodies specific for beta-amyloid by vaccination of patients with Alzheimer disease.

To characterize antibodies produced in humans in response to Aβ42 vaccination, we carried out immunohistochemical examinations of the brains of both transgenic mice and human patients with β-amyloid pathology. We collected sera from patients with Alzheimer disease who received a primary injection of pre-aggregated Aβ42 followed by one booster injection in a placebo-controlled study. Antibodies in immune sera recognized β-amyloid plaques, diffuse Aβ deposits and vascular β-amyloid in brain blood vessels. The antibodies did not cross-react with native full-length β-amyloid precursor protein or its physiological derivatives, including soluble Aβ42. These findings indicate that vaccination of AD patients with Aβ42 induces antibodies that have a high degree of selectivity for the pathogenic target structures. Whether vaccination to produce antibodies against β-amyloid will halt the cognitive decline in AD will depend upon clinical assessments over time.

The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor

The physiological functions of the beta-amyloid precursor protein (APP) may include nuclear signaling. To characterize the role of the APP adaptor proteins Fe65, Jip1b, X11α (MINT1) and the chromatin-associated protein Tip60, we analyzed their interactions by confocal microscopy and co-immunoprecipitations. AICD corresponding to S3-cleaved APP bound to Fe65 that transported it to nuclei and docked it to Tip60. These proteins formed AICD-Fe65-Tip60 (AFT) complexes that were concentrated in spherical nuclear spots. γ-Secretase inhibitors prevented AFT-complex formation with AICD derived from full-length APP. The APP adaptor protein Jip1b also transported AICD to nuclei and docked it to Tip60, but AICD-Jip1b-Tip60 (AJT) complexes had different, speckle-like morphology. By contrast, X11α trapped AICD in the cytosol. Induced AICD expression identified the APP-effector genes APP, BACE, Tip60, GSK3β and KAI1, but not the Notch-effector gene Hes1 as transcriptional targets. These data establish a role for APP in nuclear signaling, and they suggest that therapeutic strategies designed to modulate the cleavage of APP affect AICD-dependent signaling.

Intracellular Aβ and cognitive deficits precede β-amyloid deposition in transgenic arcAβ mice

The brain pathology of Alzheimer’s disease is characterized by abnormally aggregated A in extracellular -amyloid plaques and along blood vessel walls, but the relation to intracellular A remains unclear. To address the role of intracellular A deposition in vivo ,w e expressed human APP with the combined Swedish and Arctic mutations in mice (arcA mice). Intracellular punctate deposits of A occurred concomitantly with robust cognitive impairments at the age of 6 months before the onset of -amyloid plaque formation and cerebral amyloid angiopathy. -Amyloid plaques from arcA mice had distinct dense-core morphologies with blood vessels appearing as seeding origins, suggesting reduced clearance of A across blood vessels in arcA mice. The co-incidence of intracellular A deposits with behavioral deficits support an early role of intracellular A in the pathophysiological cascade leading to -amyloid formation and functional impairment.

Amyloid-β immunisation for Alzheimer’s disease

Alzheimers disease is the main cause of dementia in elderly people and is becoming an ever greater problem as societies worldwide age. Treatments that stop or at least effectively modify disease course do not yet exist. In Alzheimers disease, the conversion of the amyloid-beta peptide (Abeta) from a physiological water-soluble monomeric form into neurotoxic oligomeric and fibrillar forms rich in stable beta-sheet conformations is an important event. The most toxic forms of Abeta are thought to be oligomers, and dimers might be the smallest neurotoxic species. Numerous immunological approaches that prevent the conversion of the normal precursor protein into pathological forms or that accelerate clearance are in development. More than ten new approaches to active and passive immunotherapy are under investigation in clinical trials with the aim of producing safe methods for immunological therapy and prevention. A delicate balance between immunological clearance of an endogenous protein with acquired toxic properties and the induction of an autoimmune reaction must be found.

Aβ Oligomer-Mediated Long-Term Potentiation Impairment Involves Protein Phosphatase 1-Dependent Mechanisms

Amyloid β (Aβ) oligomers are derived from proteolytic cleavage of amyloid precursor protein (APP) and can impair memory and hippocampal long-term potentiation (LTP) in vivo and in vitro. They are recognized as the primary neurotoxic agents in Alzheimers disease. The mechanisms underlying such toxicity on synaptic functions are complex and not fully understood. Here, we provide the first evidence that these mechanisms involve protein phosphatase 1 (PP1). Using a novel transgenic mouse model expressing human APP with the Swedish and Arctic mutations that render Aβ more prone to form oligomers (arcAβ mice), we show that the LTP impairment induced by Aβ oligomers can be fully reversed by PP1 inhibition in vitro. We further demonstrate that the genetic inhibition of endogenous PP1 in vivo confers resistance to Aβ oligomer-mediated toxicity and preserves LTP. Overall, these results reveal that PP1 is a key player in the mechanisms of AD pathology.

Nuclear signaling by the APP intracellular domain occurs predominantly through the amyloidogenic processing pathway.

Proteolytic processing of the amyloid precursor protein (APP) occurs via two alternative pathways, localized to different subcellular compartments, which result in functionally distinct outcomes. Cleavage by a β-γ sequence generates the Aβ peptide that plays a central role in Alzheimers disease. In the case of α-γ cleavage, a secreted neurotrophic molecule is generated and the Aβ peptide cleaved and destroyed. In both cases, a cytosolic APP intracellular domain (AICD) is generated. We have previously shown that coexpression of APP with the APP-binding protein Fe65 and the histone acetyltransferase Tip60 results in the formation of nuclear complexes (termed AFT complexes), which localize to transcription sites. We now show that blocking endocytosis or the pharmacological or genetic inhibition of the endosomal β-cleavage pathway reduces translocation of AICD to these nuclear AFT complexes. AICD signaling further depends on active transport along microtubules and can be modulated by interference with both anterograde and retrograde transport systems. Nuclear signaling by endogenous AICD in primary neurons could similarly be blocked by inhibiting β-cleavage but not by α-cleavage inhibition. This suggests that amyloidogenic cleavage, despite representing the minor cleavage pathway of APP, is predominantly responsible for AICD-mediated nuclear signaling.

Co-localization of the amyloid precursor protein and Notch intracellular domains in nuclear transcription factories.

The beta-amyloid precursor protein (APP) plays a major role in Alzheimers disease. The APP intracellular domain (AICD), together with Fe65 and Tip60, localizes to spherical nuclear AFT complexes, which may represent sites of transcription. Despite a lack of co-localization with several described nuclear compartments, we have identified a close apposition between AFT complexes and splicing speckles, Cajal bodies and PML bodies. Live imaging revealed that AFT complexes were highly mobile within nuclei and following pharmacological inhibition of transcription fused into larger assemblies. We have previously shown that AICD regulates the expression of its own precursor APP. In support of our earlier findings, transfection of APP promoter plasmids as substrates resulted in cytosolic AFT complex formation at labeled APP promoter plasmids. In addition, identification of chromosomal APP or KAI1 gene loci by fluorescence in situ hybridization showed their close association with nuclear AFT complexes. The transcriptional activator Notch intracellular domain (NICD) localized to the same nuclear spots as occupied by AFT complexes suggesting that these nuclear compartments correspond to transcription factories. Fe65 and Tip60 also co-localized with APP in the neurites of primary neurons. Pre-assembled AFT complexes may serve to assist fast nuclear signaling upon endoproteolytic APP cleavage.

Cholesterol 25-Hydroxylase on Chromosome 10q Is a Susceptibility Gene for Sporadic Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common cause of dementia. It is characterized by β-amyloid (Aβ) plaques, neurofibrillary tangles and the degeneration of specifically vulnerable brain neurons. We observed high expression of the cholesterol 25-hydroxylase (CH25H) gene in specifically vulnerable brain regions of AD patients. CH25H maps to a region within 10q23 that has been previously linked to sporadic AD. Sequencing of the 5′ region of CH25H revealed three common haplotypes, CH25Hχ2, CH25Hχ3 and CH25Hχ4; CSF levels of the cholesterol precursor lathosterol were higher in carriers of the CH25Hχ4 haplotype. In 1,282 patients with AD and 1,312 healthy control subjects from five independent populations, a common variation in the vicinity of CH25H was significantly associated with the risk for sporadic AD (p = 0.006). Quantitative neuropathology of brains from elderly non-demented subjects showed brain Aβ deposits in carriers of CH25Hχ4 and CH25Hχ3 haplotypes, whereas no Aβ deposits were present in CH25Hχ2 carriers. Together, these results are compatible with a role of CH25Hχ4 as a putative susceptibility factor for sporadic AD; they may explain part of the linkage of chromosome 10 markers with sporadic AD, and they suggest the possibility that CH25H polymorphisms are associated with different rates of brain Aβ deposition.

Interactome of the Amyloid Precursor Protein APP in Brain Reveals a Protein Network Involved in Synaptic Vesicle Turnover and a Close Association with Synaptotagmin-1

Knowledge of the protein networks interacting with the amyloid precursor protein (APP) in vivo can shed light on the physiological function of APP. To date, most proteins interacting with the APP intracellular domain (AICD) have been identified by Yeast Two Hybrid screens which only detect direct interaction partners. We used a proteomics-based approach by biochemically isolating tagged APP from the brains of transgenic mice and subjecting the affinity-purified complex to mass spectrometric (MS) analysis. Using two different quantitative MS approaches, we compared the protein composition of affinity-purified samples isolated from wild-type mice versus transgenic mice expressing tagged APP. This enabled us to assess truly enriched proteins in the transgenic sample and yielded an overlapping set of proteins containing the major proteins involved in synaptic vesicle endo- and exocytosis. Confocal microscopy analyses of cotransfected primary neurons showed colocalization of APP with synaptic vesicle proteins in vesicular structures throughout the neurites. We analyzed the interaction of APP with these proteins using pulldown experiments from transgenic mice or cotransfected cells followed by Western blotting. Synaptotagmin-1 (Stg1), a resident synaptic vesicle protein, was found to directly bind to APP. We fused Citrine and Cerulean to APP and the candidate proteins and measured fluorescence resonance energy transfer (FRET) in differentiated SH-SY5Y cells. Differentially tagged APPs showed clear sensitized FRET emission, in line with the described dimerization of APP. Among the candidate APP-interacting proteins, again only Stg1 was in close proximity to APP. Our results strongly argue for a function of APP in synaptic vesicle turnover in vivo. Thus, in addition to the APP cleavage product Aβ, which influences synaptic transmission at the postsynapse, APP interacts with the calcium sensor of synaptic vesicles and might thus play a role in the regulation of synaptic vesicle exocytosis.