Constitutive and regulated α-secretase cleavage of Alzheimer’s amyloid precursor protein by a disintegrin metalloprotease

Amyloid β peptide (Aβ), the principal proteinaceous component of amyloid plaques in brains of Alzheimer’s disease patients, is derived by proteolytic cleavage of the amyloid precursor protein (APP). Proteolytic cleavage of APP by a putative α-secretase within the Aβ sequence precludes the formation of the amyloidogenic peptides and leads to the release of soluble APPsα into the medium. By overexpression of a disintegrin and metalloprotease (ADAM), classified as ADAM 10, in HEK 293 cells, basal and protein kinase C-stimulated α-secretase activity was increased severalfold. The proteolytically activated form of ADAM 10 was localized by cell surface biotinylation in the plasma membrane, but the majority of the proenzyme was found in the Golgi. These results support the view that APP is cleaved both at the cell surface and along the secretory pathway. Endogenous α-secretase activity was inhibited by a dominant negative form of ADAM 10 with a point mutation in the zinc binding site. Studies with purified ADAM 10 and Aβ fragments confirm the correct α-secretase cleavage site and demonstrate a dependence on the substrate’s conformation. Our results provide evidence that ADAM 10 has α-secretase activity and many properties expected for the proteolytic processing of APP. Increases of its expression and activity might be beneficial for the treatment of Alzheimer’s disease.

Axon pathology in Parkinson’s disease and Lewy body dementia hippocampus contains α-, β-, and γ-synuclein

Pathogenic α-synuclein (αS) gene mutations occur in rare familial Parkinson’s disease (PD) kindreds, and wild-type αS is a major component of Lewy bodies (LBs) in sporadic PD, dementia with LBs (DLB), and the LB variant of Alzheimer’s disease, but β-synuclein (βS) and γ-synuclein (γS) have not yet been implicated in neurological disorders. Here we show that in PD and DLB, but not normal brains, antibodies to αS and βS reveal novel presynaptic axon terminal pathology in the hippocampal dentate, hilar, and CA2/3 regions, whereas antibodies to γS detect previously unrecognized axonal spheroid-like lesions in the hippocampal dentate molecular layer. The aggregation of other synaptic proteins and synaptic vesicle-like structures in the αS- and βS-labeled hilar dystrophic neurites suggests that synaptic dysfunction may result from these lesions. Our findings broaden the concept of neurodegenerative “synucleinopathies” by implicating βS and γS, in addition to αS, in the onset/progression of PD and DLB.

Propagating structure of Alzheimer’s β-amyloid(10–35) is parallel β-sheet with residues in exact register

The pathognomonic plaques of Alzheimer’s disease are composed primarily of the 39- to 43-aa β-amyloid (Aβ) peptide. Crosslinking of Aβ peptides by tissue transglutaminase (tTg) indicates that Gln15 of one peptide is proximate to Lys16 of another in aggregated Aβ. Here we report how the fibril structure is resolved by mapping interstrand distances in this core region of the Aβ peptide chain with solid-state NMR. Isotopic substitution provides the source points for measuring distances in aggregated Aβ. Peptides containing a single carbonyl 13C label at Gln15, Lys16, Leu17, or Val18 were synthesized and evaluated by NMR dipolar recoupling methods for the measurement of interpeptide distances to a resolution of 0.2 Å. Analysis of these data establish that this central core of Aβ consists of a parallel β-sheet structure in which identical residues on adjacent chains are aligned directly, i.e., in register. Our data, in conjunction with existing structural data, establish that the Aβ fibril is a hydrogen-bonded, parallel β-sheet defining the long axis of the Aβ fibril propagation.

α-Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with Lewy bodies

Lewy bodies and Lewy neurites are the defining neuropathological characteristics of Parkinson’s disease and dementia with Lewy bodies. They are made of abnormal filamentous assemblies of unknown composition. We show here that Lewy bodies and Lewy neurites from Parkinson’s disease and dementia with Lewy bodies are stained strongly by antibodies directed against amino-terminal and carboxyl-terminal sequences of α-synuclein, showing the presence of full-length or close to full-length α-synuclein. The number of α-synuclein-stained structures exceeded that immunoreactive for ubiquitin, which is currently the most sensitive marker of Lewy bodies and Lewy neurites. Staining for α-synuclein thus will replace staining for ubiquitin as the preferred method for detecting Lewy bodies and Lewy neurites. We have isolated Lewy body filaments by a method used for the extraction of paired helical filaments from Alzheimer’s disease brain. By immunoelectron microscopy, extracted filaments were labeled strongly by anti-α-synuclein antibodies. The morphologies of the 5- to 10-nm filaments and their staining characteristics suggest that extended α-synuclein molecules run parallel to the filament axis and that the filaments are polar structures. These findings indicate that α-synuclein forms the major filamentous component of Lewy bodies and Lewy neurites.

Phosphorylated α-synuclein-immunoreactive retinal neuronal elements in Parkinson's disease subjects

Visual symptoms are relatively common in Parkinson's disease (PD) and optical coherence tomography has indicated possible retinal thinning. Accumulation of aggregated α-synuclein is thought to be a central pathogenic event in the PD brain but there have not as yet been reports of retinal synucleinopathy. Retinal wholemounts were prepared from subjects with a primary clinicopathological diagnosis of PD (N = 9), dementia with Lewy bodies (DLB; N = 3), Alzheimer's disease (N = 3), progressive supranuclear palsy (N = 2) as well as elderly normal control subjects (N = 4). These were immunohistochemically stained with an antibody against α-synuclein phosphorylated at serine 129, which is a specific molecular marker of synucleinopathy. Phosphorylated α-synuclein-immunoreactive (p-syn IR) nerve fibers were present in 7/9 PD subjects and in 1/3 DLB subjects; these were sparsely distributed and superficially located near or at the inner retinal surface. The fibers were either long and straight or branching, often with multiple en-passant varicosities along their length. The straight fibers most often had an orientation that was radial with respect to the optic disk. Together, these features are suggestive of either retinopetal/centrifugal fibers or of ganglion cell axons. In one PD subject there were sparse p-syn IR neuronal cell bodies with dendritic morphology suggestive of G19 retinal ganglion cells or intrinsically photosensitive ganglion cells. There were no stained nerve fibers or other specific staining in any of the non-PD or non-DLB subjects. It is possible that at least some of the observed visual function impairments in PD subjects might be due to α-synucleinopathy.

Prevalence of mixed neuropathologies in autopsied older adults and associations with clinical disease progression

Thesis (Ph.D.)--University of Washington, 2016-06

A novel screen for nuclear mitochondrial gene associations with Parkinson's disease

Genetic factors play an important role in the aetiology of Parkinson's disease (PD). We have screened nuclear genes encoding subunits of mitochondrial complex I for associations between single nucleotide polymorphisms (SNPs) and PD. Abnormal functioning of complex I is well documented in human PD. Moreover, toxicological inhibition of complex I can lead to parkinsonism in animals. Thus, commonly occurring variants in these genes could potentially influence complex I function and the risk of developing PD. A sub-set of 70 potential SNPs in 31 nuclear complex I genes were selected and association analysis was performed on 306 PD patients plus 321 unaffected control subjects. Genotyping was performed using the DASH method. There was no evidence that the examined SNPs were significant genetic risk factors for PD, although this initial screen could not exclude the possibility that other disease-influencing variations exist within these genes.

Spatial correlations between beta-amyloid (Abeta) deposits and blood vessels in early-onset Alzheimer's disease

In cases of late-onset Alzheimer’s disease (AD), there is a spatial correlation between the classsic ‘cored’ type of Beta-amyloid (Abeta) deposit and the large vertically penetrating arterioles in the cerebral cortex suggesting that blood vessels are involved in the pathogenesis of the classic deposits. In this chapter, the spatial correlations between the diffuse, primitive, and classic Abeta deposits and blood vessels were studied in 10 cases of early-onset AD in the age range 40 – 65 years. Sections of frontal cortex were immunostained with antibodies against Abeta?and with collagen IV to reveal the Abeta deposits and blood vessel profiles. In the early-onset cases as a whole, all types of Abeta? deposit and blood vessel profiles were distributed in clusters. There was a positive spatial correlation between the clusters of the diffuse Abeta deposits and the larger (>10µm) and smaller diameter (<10?m) blood vessel profiles in one and three cases respectively. The primitive and classic Abeta deposits were spatially correlated with larger and smaller blood vessels both in three and four cases respectively. Spatial correlations between the Abeta deposits and blood vessels may be more prevalent in cases expressing amyloid precursor protein (APP) than presenilin 1 (PSEN1) mutations. Apolipoprotein E (Apo E) genotype of the patient did not appear to influence the spatial correlation with blood vessel profiles. The data suggest that the larger diameter blood vessels are less important in the pathogenesis of the classic Abeta deposits in early-onset compared with late-onset AD.

The spatial patterns of beta-amyloid (Abeta) deposits and neurofibrillary tangles (NFT) in late-onset sporadic Alzheimer's disease

The spatial patterns of beta-amyloid (Abeta) deposits and neurofibrillary tangles (NFT) were studied in areas of the cerebral cortex in 16 patients with the late-onset, sporadic form of Alzheimer’s disease (AD). Diffuse, primitive, and classic Abeta deposits and NFT were aggregated into clusters; the clusters being regularly distributed parallel to the pia mater in many areas. In a significant proportion of regions, the sizes of the regularly distributed clusters approximated to those of the cells of origin of the cortico-cortical projections. The diffuse and primitive Abeta deposits exhibited a similar range of spatial patterns but the classic Abeta deposits occurred less frequently in large clusters >6400microm. In addition, the NFT often occurred in larger regularly distributed clusters than the Abeta deposits. The location, size, and distribution of the clusters of Abeta deposits and NFT supports the hypothesis that AD is a 'disconnection syndrome' in which degeneration of specific cortico-cortical and cortico-hippocampal pathways results in synaptic disconnection and the formation of clusters of NFT and Abeta deposits.

Spatial correlations between beta-amyloid (A beta) deposits and blood vessels in familial Alzheimer's disease

In sporadic Alzheimer’s disease (SAD), the classic (‘dense-cored’) ß-amyloid (Aß) deposits are aggregated around the larger blood vessels in the upper laminae of the cerebral cortex. To determine whether a similar relationship exists in familial AD (FAD), the spatial correlations between the diffuse, primitive, and classic ß-amyloid (Aß deposits and blood vessels were studied in ten FAD cases including cases linked to amyloid precursor protein (APP) and presenilin (PSEN) gene mutations and expressing apolipoprotein E (apo E) allele E4. Sections of frontal cortex were immunolabelled with antibodies against Aß and with collagen IV to reveal the Aß deposits and blood vessel profiles. In the FAD cases as a whole, Aßdeposits were distributed in clusters. There was a positive spatial correlation between the clusters of the diffuse Aßdeposits and the larger (>10 µm) and smaller diameter (<10 µm) blood vessels in one and three cases respectively. The primitive Aß deposits were spatially correlated with larger and smaller blood vessels each in four cases and the classic deposits in three and four cases respectively. Apo E genotype of the patient did not influence spatial correlation with blood vessels. Hence, spatial correlations between the classic deposits and larger diameter blood vessels were significantly less frequent in FAD compared with SAD. It was concluded that both Aß deposit morphology and AD subtype determine spatial correlations with blood vessels in AD.

Neuropathological differences between areas B17 and B18: implications for visual evoked responses in Alzheimer's disease

The density of senile plaques (SP) and neurofibrillary tangles (NFT) was estimated at post-mortem in areas B17 and B18 of the visual cortex in 18 Alzheimer’s disease (AD) cases which varied in disease onset and duration. The density of SP in B17 and NFT in B17 and B18 declined significantly with age at death of the patient. The density of SP and NFT was greater in B18 than B17 but only in cases of earlier onset and shorter duration. The pathological differences between B17 and B18 could explain the visual evoked responses (VER) that have been reported in AD. However, the differences were small, and changes in the afferent pathways remain the most likely explanation for the VER in AD. © 1994 S. Karger AG, Basel.

Ocular and systemic vascular dysfunction in neurodegenerative disease

The important role played by vascular factors in the pathogenesis of neurodegenerative disease has been increasingly realised over recent years. The nature and impact of ocular and systemic vascular dysfunction in the pathogenesis of comparable neurodegenerative diseases such as glaucoma and Alzheimer’s disease (AD) has however never been fully explored. The aim of this thesis was therefore to investigate the presence of macro- and micro-vascular alterations in both glaucoma and AD and to explore the relationships between these two chronic, slowly progressive neurodegenerative diseases. The principle sections and findings of this work were: 1. Is the eye a window to the brain? Retinal vascular dysfunction in Alzheimer’s disease · Mild newly diagnosed AD patients demonstrated ocular vascular dysfunction, in the form of an altered retinal vascular response to flicker light, which correlated with their degree of cognitive impairment. 2. Ocular and systemic vascular abnormalities in newly diagnosed normal tension glaucoma (NTG) patients · NTG patients demonstrated an altered retinal arterial constriction response to flicker light along with an increased systemic arterial stiffness and carotid artery intima-media thickness (IMT). These findings were not replicated by healthy age matched controls. 3. Ocular vascular dysregulation in AD compares to both POAG and NTG · AD patients demonstrated altered retinal arterial reactivity to flicker light which was comparable to that of POAG patients and altered baseline venous reactivity which was comparable to that of NTG patients. Neither alteration was replicated by healthy controls. 4. POAG and NTG: two separate diseases or one continuous entity? The vascular perspective · POAG and NTG patients demonstrated comparable alterations in nocturnal systolic blood pressure (SBP) variability, ocular perfusion pressure, retinal vascular reactivity, systemic arterial stiffness and carotid IMT. · Nocturnal SBP variability was found to correlate with both retinal artery baseline diameter fluctuation and carotid IMT across the glaucoma groups.

The visual cortex in Alzheimer disease:laminar distribution of the pathological changes in visual areas V1 and V2

Alzheimer’s disease (AD) is an important neurodegenerative disorder causing visual problems in the elderly population. The pathology of AD includes the deposition in the brain of abnormal aggregates of ?-amyloid (A?) in the form of senile plaques (SP) and abnormally phosphorylated tau in the form of neurofibrillary tangles (NFT). A variety of visual problems have been reported in patients with AD including loss of visual acuity (VA), colour vision and visual fields; changes in pupillary responses to mydriatics, defects in fixation and in smooth and saccadic eye movements; changes in contrast sensitivity and in visual evoked potentials (VEP); and disturbances in complex visual tasks such as reading, visuospatial function, and in the naming and identification of objects. In addition, pathological changes have been observed to affect the eye, visual pathway, and visual cortex in AD. To better understand degeneration of the visual cortex in AD, the laminar distribution of the SP and NFT was studied in visual areas V1 and V2 in 18 cases of AD which varied in disease onset and duration. In area V1, the mean density of SP and NFT reached a maximum in lamina III and in laminae II and III respectively. In V2, mean SP density was maximal in laminae III and IV and NFT density in laminae II and III. The densities of SP in laminae I of V1 and NFT in lamina IV of V2 were negatively correlated with patient age. No significant correlations were observed in any cortical lamina between the density of NFT and disease onset or duration. However, in area V2, the densities of SP in lamina II and lamina V were negatively correlated with disease duration and disease onset respectively. In addition, there were several positive correlations between the densities of SP and NFT in V1 with those in area V2. The data suggest: (1) NFT pathology is greater in area V2 than V1, (2) laminae II/III of V1 and V2 are most affected by the pathology, (3) the formation of SP and NFT in V1 and V2 are interconnected, and (4) the pathology may spread between visual areas via the feed-forward short cortico-cortical connections.