Abstract Body

Aims

Aβ peptides differing at N- and C-termini coincide in brain structures affected by Alzheimer's Disease (AD). Their aggregation is related to the onset of AD. These peptides are co-present with physiological metal cations including Cu²⁺ and Zn²⁺. These metal ions form complexes with Aβ species depending on their N-terminal sequences. Our aim was to find out how N-terminal truncation and Cu²⁺ and Zn²⁺ presence affect the aggregation behaviour of the most abundant Aβ species, Aβ_1-x and Aβ_4-x, where x = 40, 42, in order to obtain a more realistic model of the Aβ aggregation process.

Methods

The aggregation process was studied by the time course of Aβ fibril formation using ThT fluorescence and the analysis of moprphology of the formed aggregates by TEM. The kinetic studies were aided by turbidimetric measurements. Metal complex formation was followed using circular dichroism spectroscopy and NMR. The kinetic profiles were subjected to mathematical analysis in order to extract the quantitative parameters of the aggregation process.

Results

In both peptide types substoichiometric metal cations accelerated aggregation, while superstoichiometric ones inhibited the fibrillation. The addition of either Aβ_4-x or substoichiometric cations affected the aggregation profile of Aβ_1-x similarly and yielded shorter and thicker fibrils; amorphous aggregates were formed at the metal molar excess. Analogous effects exerted by Aβ_4-x and substoichiometric cations can be attributed to the appearance of positive charge at Aβ N-terminus, caused by either complexation or N-truncation.

Conclusions

We found an electrostatic principle for enhanced/delayed seeding of Aβ aggregation which can be used to control/inhibit this process.

Abstract Body

Compelling data support a contemporary version of the amyloid cascade hypothesis (ACH) as a valid framework both for understanding AD pathogenesis and the development of disease modifying therapeutics. However, Aβ aggregate accumulation may not be sufficiently toxic to induce downstream neurodegeneration unless accompanied by accumulation of other proteins (amyloid associated proteins (AAPs) in the plaque. Indeed, AAPs could help trigger the neurodegenerative phase of AD, accounting for the long delay between onset of Aβ deposition and neurodegeneration in humans. We have used state of the art proteomics to identify a large number of candidate AAPs that are increased both in AD and mouse models of Aβ deposition. Many of these AAPs have known or inferred cell-signaling functions. Further, for some candidate AAPs there is either previous data demonstrating that they are AD AAPs or we have generated novel data showing accumulation in senile plaques. Finally, as shown by others for the AAPs, ApoE and clusterin, we find that expression of select AAPs (midkine, pleiotrophin) modulates amyloid deposition and promote dramatic shifts to CAA. These data reveal the complexity of the proteinopathy in AD, and provide the field a novel framework in which to conduct future studies.

Abstract Body

Recent genome-wide association studies identified the angiotensin-converting enzyme gene (ACE) as an Alzheimer’s disease (AD) risk locus. However, the pathogenic mechanism by which ACE causes AD is unknown. Using whole-genome sequencing, we identified rare ACE coding variants in AD families. We chose to investigate one, ACE1 R1279Q, by generating knockin (KI) mice using CRSPR/Cas9 genome editing. We characterized KI mice with behavioral, electrophysiological, biochemical, cell biological, and histological methods. Similar to AD, ACE1 was increased in neurons, but not microglia or astrocytes, of KI brains, which became elevated further with age. Angiotensin II (angII) and angII receptor AT1R signaling were also increased in KI brains. Autosomal dominant neurodegeneration and neuroinflammation occurred with aging in KI hippocampus, which were absent in the cortex and cerebellum. Female KI mice exhibited greater hippocampal electroencephalograph disruption and memory impairment compared to males. ACE variant effects were more pronounced in female KI mice, suggesting a mechanism for higher AD risk in women. Hippocampal neurodegeneration was completely rescued by treatment with brain-penetrant drugs that inhibit ACE1 and AT1R. Although ACE variant-induced neurodegeneration did not depend on β-amyloid (Aβ) pathology, amyloidosis in 5XFAD mice crossed to KI mice accelerated neurodegeneration and neuroinflammation, whereas Aβ deposition was unchanged. KI mice had normal blood pressure and cerebrovascular functions. Our findings strongly suggest that increased ACE1/angII signaling causes aging-dependent, Aβ-accelerated selective hippocampal neuron vulnerability and female susceptibility, hallmarks of AD that have hitherto been enigmatic. We conclude that repurposed brain-penetrant ACE inhibitors and AT1R blockers may protect against AD.

Aims

Recent genome-wide association studies identified the angiotensin-converting enzyme gene (ACE) as an Alzheimer’s disease (AD) risk locus. However, the pathogenic mechanism by which ACE causes AD is unknown.

Methods

Using whole-genome sequencing, we identified rare ACE coding variants in AD families. We chose to investigate one, ACE1 R1279Q, by generating knockin (KI) mice using CRSPR/Cas9 genome editing. We characterized KI mice with behavioral, electrophysiological, biochemical, cell biological, and histological methods.

Results

Similar to AD, ACE1 was increased in neurons, but not microglia or astrocytes, of KI brains, which became elevated further with age. Angiotensin II (angII) and angII receptor AT1R signaling were also increased in KI brains. Autosomal dominant neurodegeneration and neuroinflammation occurred with aging in KI hippocampus, which were absent in the cortex and cerebellum. Female KI mice exhibited greater hippocampal electroencephalograph disruption and memory impairment compared to males. ACE variant effects were more pronounced in female KI mice, suggesting a mechanism for higher AD risk in women. Hippocampal neurodegeneration was completely rescued by treatment with brain-penetrant drugs that inhibit ACE1 and AT1R. Although ACE variant-induced neurodegeneration did not depend on β-amyloid (Aβ) pathology, amyloidosis in 5XFAD mice crossed to KI mice accelerated neurodegeneration and neuroinflammation, whereas Aβ deposition was unchanged. KI mice had normal blood pressure and cerebrovascular functions.

Conclusions

Our findings strongly suggest that increased ACE1/angII signaling causes aging-dependent, Aβ-accelerated selective hippocampal neuron vulnerability and female susceptibility, hallmarks of AD that have hitherto been enigmatic. We conclude that repurposed brain-penetrant ACE inhibitors and AT1R blockers may protect against AD.

Abstract Body

This lecture will present actuarial methods for diagnosing neuropsychological features of mild cognitive impairment (MCI) and objectively-defined subtle cognitive deficits (Obj-SCD) of prodromal Alzheimer’s disease (AD). Seeking to refine diagnostic and prediction models, in a series of studies we have compared conventional criteria used to diagnose MCI with our actuarial neuropsychological methods. Results from these studies suggest that conventional criteria are susceptible to false positive and false negative diagnostic errors, whereas MCI participants diagnosed via neuropsychological criteria yield specific cognitive profiles, significant biomarker associations, more stable diagnoses, and greater percentages who progress to dementia than conventional MCI diagnostic criteria. We further extend this actuarial method to support refinement of research criteria for Obj-SCD in preclinical AD diagnosis. This lecture will thus reflect research and clinical advances in identifying cognitive features of MCI and preclinical AD, examining its biomarker signatures, and offer new possibilities for improving diagnostic precision. The evidence reviewed in this lecture concludes that a multi-faceted approach that integrates neuropsychological and biomarker assessments will likely be needed to characterize the preclinical phase of AD and ends with the suggestion that neuropsychological assessment provides a central and non-interchangeable role in the diagnosis of the older adult suspected of dementia due to AD, and that cognitive measures are among the best predictors of the initial symptomatic stages of an evolving dementia.

Aims

Cerebrovascular dysfunction is one of the earliest events in Alzheimer’s disease (AD) pathogenesis. While endothelial cells are crucial to maintain neurovascular unit function, amyloid beta (Aβ) vascular deposits as well as chronic cardiovascular (CV) risk factors are known to impair endothelial, blood brain barrier (BBB), and neurovascular health. However, the mechanisms responsible for these effects are still poorly understood, and strategies to prevent neurovascular unit dysfunction are needed.

Methods

Our studies employed human cerebral microvascular endothelial cells in culture and mouse models of vascular amyloidosis to clarify mitochondrial and cellular mechanisms by which amyloid induces BBB and neurovascular unit stress, the contribution of CV risk factors, and possible therapeutic avenues.

Results

We found that Aβ challenge induced death receptors activation with mitochondrial metabolic and respiratory deficits, triggering apoptotic pathways in endothelial cells, BBB permeability and angiogenesis defects. Hyperhomocysteinemia (HHCy) potentiated the effects of Aβ on BBB dysfunction. The deleterious effects of Aβ were counteracted by carbonic anhydrase inhibitors (CAIs) in both cell and animal models of cerebral amyloid angiopathy (CAA). Our studies in a mouse model of vascular amyloidosis showed that cerebrovascular degeneration, neuroinflammation, vascular and immune cell-specific caspase activation, and memory impairment were all diminished by CAIs.

Conclusions

Our studies are clarifying mitochondrial and cell stress mechanisms contributing to vascular cell dysfunction triggered by amyloid aggregates, identifying the contributions of CV risk factors such as HHcy, and pioneering the study of CAs and their inhibitors in AD and CAA.