Browsing by Author "Walter, Jochen"

Rare coding variants in the triggering receptor expressed on myeloid cells-2 (TREM2) gene have been associated with Alzheimer disease (AD) and homozygous TREM2 loss-of-function variants have been reported in families with monogenic frontotemporal-like dementia with/without bone abnormalities. In a whole-exome sequencing study of a family with probable AD-type dementia without pathogenic variants in known autosomal dominant dementia disease genes and negative for the apolipoprotein E (APOE) ε4 allele, we identified an extremely rare TREM2 coding variant, that is, a glycine-to-tryptophan substitution at amino acid position 145 (NM_018965.3:c.433G>T/p.[Gly145Trp]). This alteration is found in only 1 of 251,150 control alleles in gnomAD. It was present in both severely affected as well as in another putatively affected and one 61 years old as yet unaffected family member suggesting incomplete penetrance and/or a variable age of onset. Gly145 maps to an intrinsically disordered region (IDR) of TREM2 between the immunoglobulin-like and transmembrane domain. Subsequent cellular studies showed that the variant led to IDR shortening and structural changes of the mutant protein resulting in an impairment of cellular responses upon receptor activation. Our results, suggest that a p.(Gly145Trp)-induced structural disturbance and functional impairment of TREM2 may contribute to the pathogenesis of an AD-like form of dementia.

The progressive accumulation of extracellular amyloid plaques in the brain is a common hallmark of Alzheimer's disease (AD). We recently identified a novel species of A beta phosphorylated at serine residue 8 with increased propensity to form toxic aggregates as compared to non-phosphorylated species. The age-dependent analysis of A beta depositions using novel monoclonal phosphorylation-state specific antibodies revealed that phosphorylated A beta variants accumulate first inside of neurons in a mouse model of AD already at 2 month of age. At higher ages, phosphorylated A beta is also abundantly detected in extracellular plaques. Besides a large overlap in the spatiotemporal deposition of phosphorylated and non-phosphorylated A beta species, fractionized extraction of A beta from brains revealed an increased accumulation of phosphorylated A beta in oligomeric assemblies as compared to non-phosphorylated A beta in vivo. Thus, phosphorylated A beta could represent an important species in the formation and stabilization of neurotoxic aggregates, and might be targeted for AD therapy and diagnosis.

The beta-amyloid precursor protein (APP) represents a type I transmembrane glycoprotein that is ubiquitously expressed. In the brain, it is a key player in the molecular pathogenesis of Alzheimer disease. Its physiological function is however less well understood. Previous studies showed that APP is up-regulated in prostate, colon, pancreatic tumor, and oral squamous cell carcinoma. In this study, we show that APP has an essential role in growth control of pancreatic and colon cancer. Abundant APP staining was found in human pancreatic adenocarcinoma and colon cancer tissue. Interestingly, treating pancreatic and colon cancer cells with valproic acid (VPA, 2-propylpentanoic acid), a known histone deacetylase (HDAC) inhibitor, leads to up-regulation of GRP78, an endoplasmic reticulum chaperone immunoglobulin-binding protein. GRP78 is involved in APP maturation and inhibition of tumor cell growth by down-regulation of APP and secreted soluble APP alpha. Trichostatin A, a pan-HDAC inhibitor, also lowered APP and increased GRP78 levels. In contrast, treating cells with valpromide, a VPA derivative lacking HDAC inhibitory properties, had no effect on APP levels. VPA did not modify the level of epidermal growth factor receptor, another type I transmembrane protein, and APLP2, a member of the APP family, demonstrating the specificity of the VPA effect on APP. Small interfering RNA-mediated knockdown of APP also resulted in significantly decreased cell growth. Based on these observations, the data suggest that APP downregulation via HDAC inhibition provides a novel mechanism for pancreatic and colon cancer therapy.

N-terminally truncated A beta peptides starting with pyroglutamate (A beta pE3) represent a major fraction of all A beta peptides in the brain of Alzheimer disease (AD) patients. A beta pE3 has a higher aggregation propensity and stability and shows increased toxicity compared with full-length A beta. In the present work, we generated a novel monoclonal antibody (9D5) that selectively recognizes oligomeric assemblies of A beta pE3 and studied the potential involvement of oligomeric A beta pE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost nondetectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal and blood vessel staining was observed. Using a novel sandwich ELISA significantly decreased levels of oligomers in plasma samples from patients with AD compared with healthy controls were identified. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall A beta plaque load and A beta pE3 levels, and normalized behavioral deficits. These data indicate that 9D5 is a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight A beta pE3 oligomers.

Locus ceruleus (LC)-supplied norepinephrine (NE) suppresses neuroinflammation in the brain. To elucidate the effect of LC degeneration and subsequent NE deficiency on Alzheimer's disease pathology, we evaluated NE effects on microglial key functions. NE stimulation of mouse microglia suppressed A beta-induced cytokine and chemokine production and increased microglial migration and phagocytosis of A beta. Induced degeneration of the locus ceruleus increased expression of inflammatory mediators in APP-transgenic mice and resulted in elevated A beta deposition. In vivo laser microscopy confirmed a reduced recruitment of microglia to A beta plaque sites and impaired microglial A beta phagocytosis in NE-depleted APP-transgenic mice. Supplying the mice the norepinephrine precursor L-threo-DOPS restored microglial functions in NE-depleted mice. This indicates that decrease of NE in locus ceruleus projection areas facilitates the inflammatory reaction of microglial cells in AD and impairs microglial migration and phagocytosis, thereby contributing to reduced A beta clearance. Consequently, therapies targeting microglial phagocytosis should be tested under NE depletion.

Neurodegeneration is characterized by the ubiquitous presence of modifications in protein deposits. Despite their potential significance in the initiation and progression of neurodegenerative diseases, the effects of posttranslational modifications on the molecular properties of protein aggregates are largely unknown. Here, we study the Alzheimer disease-related amyloid-beta (A beta) peptide and investigate how phosphorylation at serine 8 affects the structure of A beta aggregates. Serine 8 is shown to be located in a region of high conformational flexibility in monomeric A beta, which upon phosphorylation undergoes changes in local conformational dynamics. Using hydrogen-deuterium exchange NMR and fluorescence quenching techniques, we demonstrate that A beta phosphorylation at serine 8 causes structural changes in the N-terminal region of A beta aggregates in favor of less compact conformations. Structural changes induced by serine 8 phosphorylation can provide a mechanistic link between phosphorylation and other biological events that involve the N-terminal region of A beta aggregates. Our data therefore support an important role of posttranslational modifications in the structural polymorphism of amyloid aggregates and their modulatory effect on neurodegeneration.

Protein aggregation plays a crucial role in neurodegenerative diseases. A key feature of protein aggregates is their ubiquitous modification by phosphorylation. Little is known, however, about the molecular consequences of phosphorylation of protein aggregates. Here we show that phosphorylation of beta-amyloid at serine 8 increases the stability of its pathogenic aggregates against high-pressure and SDS-induced dissociation. We further demonstrate that phosphorylation results in an elevated number of hydrogen bonds at the N terminus of beta-amyloid, the region that is critically regulated by a variety of post-translational modifications. Because of the increased lifetime of phosphorylated beta-amyloid aggregates, phosphorylation can promote the spreading of beta-amyloid in Alzheimer pathogenesis. Our study suggests that regulation of the molecular stability of protein aggregates by post-translational modifications is a crucial factor for disease progression in the brain.

Aggregation and toxicity of the amyloid beta-peptide (A beta) are considered as critical events in the initiation and progression of Alzheimer's disease (AD). Recent evidence indicated that soluble oligomeric A beta assemblies exert pronounced toxicity, rather than larger fibrillar aggregates that deposit in the forms of extracellular plaques. While some rare mutations in the A beta sequence that cause early-onset AD promote the oligomerization, molecular mechanisms that induce the formation or stabilization of oligomers of the wild-type A beta remain unclear. Here, we identified an A beta variant phosphorylated at Ser26 residue (pSer26A beta) in transgenic mouse models of AD and in human brain that shows contrasting spatio-temporal distribution as compared to non-phosphorylated A beta (npA beta) or other modified A beta species. pSer26A beta is particularly abundant in intraneuronal deposits at very early stages of AD, but much less in extracellular plaques. pSer26A beta assembles into a specific oligomeric form that does not proceed further into larger fibrillar aggregates, and accumulates in characteristic intracellular compartments of granulovacuolar degeneration together with TDP-43 and phosphorylated tau. Importantly, pSer26A beta oligomers exert increased toxicity in human neurons as compared to other known A beta species. Thus, pSer26A beta could represent a critical species in the neurodegeneration during AD pathogenesis.

Pathogenesis of Alzheimer's disease (AD) is associated with aggregation of the amyloid-beta (A beta) peptide into oligomeric and fibrillar assemblies; however, little is known about the molecular basis of aggregation of A beta into distinct assembly states. Here we demonstrate that phosphorylation at serine 26 (S26) impairs A beta fibrillization while stabilizing its monomers and nontoxic soluble assemblies of nonfibrillar morphology. NMR spectroscopy and replica-exchange molecular dynamics indicate that introduction of a phosphate group or phosphomimetic at position 26 diminishes A beta's propensity to form a beta-hairpin, rigidifies the region around the modification site, and interferes with formation of a fibril-specific salt bridge between aspartic acid 23 and lysine 28. The combined data demonstrate that phosphorylation of S26 prevents a distinct conformational rearrangement that is required for progression of A beta aggregation toward fibrils and provide a basis for a possible role of phosphorylation at serine 26 in AD.