An integrative multi-omics approach reveals new central nervous system pathway alterations in Alzheimer's disease

Background Multiple pathophysiological processes have been described in Alzheimer's disease (AD). Their inter-individual variations, complex interrelations, and relevance for clinical manifestation and disease progression remain poorly understood. We hypothesize that specific molecular patterns indicating both known and yet unidentified pathway alterations are associated with distinct aspects of AD pathology. Methods We performed multi-level cerebrospinal fluid (CSF) omics in a well-characterized cohort of older adults with normal cognition, mild cognitive impairment, and mild dementia. Proteomics, metabolomics, lipidomics, one-carbon metabolism, and neuroinflammation related molecules were analyzed at single-omic level with correlation and regression approaches. Multi-omics factor analysis was used to integrate all biological levels. Identified analytes were used to construct best predictive models of the presence of AD pathology and of cognitive decline with multifactorial regression analysis. Pathway enrichment analysis identified pathway alterations in AD. Results Multi-omics integration identified five major dimensions of heterogeneity explaining the variance within the cohort and differentially associated with AD. Further analysis exposed multiple interactions between single 'omics modalities and distinct multi-omics molecular signatures differentially related to amyloid pathology, neuronal injury, and tau hyperphosphorylation. Enrichment pathway analysis revealed overrepresentation of the hemostasis, immune response, and extracellular matrix signaling pathways in association with AD. Finally, combinations of four molecules improved prediction of both AD (protein 14-3-3 zeta/delta, clusterin, interleukin-15, and transgelin-2) and cognitive decline (protein 14-3-3 zeta/delta, clusterin, cholesteryl ester 27:1 16:0 and monocyte chemoattractant protein-1). Conclusions Applying an integrative multi-omics approach we report novel molecular and pathways alterations associated with AD pathology. These findings are relevant for the development of personalized diagnosis and treatment approaches in AD.

The Gamma-Secretase-Mediated Proteolytic Processing of APP C-Terminal Fragments as a Therapeutic Target for Alzheimer's Disease

Jemila Houacine

Alzheimer's disease (AD) is a devastating neurodegenerative disorder which severely impairs cognitive functions by triggering neuronal cell death and synaptic loss, and finally leads the patients to death. Two main histopathological hallmarks can be found in the brain tissue of AD patients: the amyloid plaques composed of aggregated Amyloid-β peptides (Aβ) and the neurofibrillary tangles (NFTs) constituted of hyperphosphorylated tau protein. To explain the underlying molecular mechanisms of AD pathogenesis, the amyloid cascade hypothesis states that early accumulation of Aβ peptides (especially the Aβ42 specie identified as the most toxic one) triggers their assembly into oligomers and their subsequent extracellular aggregation into dense fibrillar deposits. The oligomeric Aβ assemblies further initiate a succession of neurotoxic events including disruptions at the synaptic level, inflammation, neuritic injury, altered calcium homeostasis, oxidative stress, and altered phosphorylation activity leading to the hyperphosphorylation of tau and its aggregation into NFTs. Finally, this cascade of neuronal deleterious effects induces cognitive dysfuntions leading to dementia. The Aβ peptides originate from the amyloidogenic processing of the amyloid precursor protein (APP), a type I membrane protein that undergoes a first cleavage by β-secretase to liberate the soluble APP domain (sAPPβ) in the extracellular space, and the membrane bound APP-C99 fragment. Then, APP-C99 is processed by the intramembrane aspartyl-protease gamma-secretase (γ-secretase) composed of four subunits (presenilin (PS), nicastrin (NCT), anterior pharynx-defective protein 1 (APH1), and presenilin enhancer 2 (PEN2)) to release the toxic Aβ peptides in the lumen, and the APP intracellular domain (AICD) in the cytosol. Alternatively, APP can undergo the non-amyloidogenic processing, in which it is first shedded by α-secretase to generate the sAPPα domain and the APP-C83 fragment. The latter is further cleaved by γ-secretase into the non toxic p3 peptide and the AICD. AD is the most frequent form of dementia in elderly humans. Despite this evidence, no treatment is currently available to prevent or cure this disease. Thus, multiple potential drugs are tested in clinical trials or are still being developed in preclinical studies. In this work, a new therapeutic approach to lower Aβ production by specifically targeting the γ-secretase-mediated processing of APP-C99 is presented. Monoclonal antibodies against APP-C99 were generated and characterized following mice immunization with the active recombinant substrate APP-C99. When tested in cells, these antibodies decreased the γ-secretase-dependent processing of APP-C99, thus lowering Aβ production. Furthermore, the intracerebroventricular injection of anti-APP-C99 antibodies in a mouse model of AD decreased total soluble Aβ levels. These results validated this new approach as a potential immunotherapeutic strategy to prevent and/or delay the neurotoxic effects caused by Aβ in AD pathogenesis. Next, the γ-secretase-mediated processing of the APP-carboxy-terminal fragments (APP-CTFs) regulating the toxic versus non-toxic pathways was further investigated with the help of in vitro activity assays. This comparative study of APP-C99 and APP-C83 processing by γ-secretase revealed that both substrates were identically processed by the enzyme, with the same AICDs being produced. In addition, the effects of familial AD (FAD) mutations in the PS subunit of highly pure and homogeneous γ-secretase complexes were also investigated, and showed a drastic loss-of-function phenotype linked to these mutants. Overall, in vitro studies of the APP-CTFs processing by γ-secretase underlined particular functional aspects of this processing, which could be related to the cellular pathways involved in AD pathogenesis such as the competition between the amyloidogenic and non-amyloidogenic pathways and the FAD-linked impaired metabolism of APP.

EPFL2011

Application of anti-amyloid beta immunization using encapsulated cell technology in mouse models of Alzheimer's disease

Vanessa Laversenne

Alzheimer's disease (AD) is the most common form of dementia in the elderly. AD is characterized by the deposition of two aggregated proteins: Amyloid beta (Aß) and hyperphosphorylated tau. Accumulations of these proteins are thought to be the signature of a pathogenic process causing the loss of neurons and synaptic connections associated with severe cognitive impairments in AD patients. Other important factors are involved in the neurodegeneration occurring in AD, such as neuroinflammation. In the present thesis work, we are investigating the effects of passive anti-Aß immunization. Passive anti-Aß immunization consists in the delivery of antibodies targeting Aß, with the aim to prevent its accumulation and promote the removal of pathological amyloid species. Although the treatment has been shown to clear amyloid pathology in the brain of AD patients, it has so far failed to prevent or slow down cognitive decline. Hence, it is important to determine the effects of anti-Aß immunization when administered either before or during Aß deposition, and explore how the treatment affects the Alzheimerâs manifestations in mouse models combining Aß and tau pathologies. In order to continuously deliver antibodies in AD mouse models, we used encapsulated cell technology (ECT) to implant myoblasts genetically engineered and secrete the recombinant mAb11 mouse IgG2a antibody directed against aggregated forms of Aß. Using this approach, we investigated the following three topics of research:

We examined the effect of a preventive administration of anti-Aß antibodies in a slowly progressing mouse model with both Aß and tau (P301L) pathologies. We demonstrated that delivery of anti-Aß antibodies before the onset of the disease dramatically reduces Aß pathology. We also found that the treatment decreases hyperphosphorylated tau accumulation and recruits microglial around Aß plaques.
We next assessed the effects of passive anti-Aß immunization in another AD mouse model overexpressing both Aß and wild-type (WT) tau. In this second study, we administered the anti-Aß antibodies when both Aß and tau pathologies were already established. In the hippocampal formation, we showed that WT tau overexpression might affect the microglial response to ongoing pathology by reducing the tight interaction of these cells with the amyloid plaques. This effect, however, is counteracted by passive anti-Aß immunization, which was found to redirect microglia towards Aß deposits. In addition, passive anti-Aß immunization decreases tau spreading throughout the hippocampus, but fails to prevent neither tau phosphorylation, nor the hippocampal degeneration induced by local tau overexpression.
In parallel, we developed a human myoblast cell line secreting anti-Aß antibodies for translation of our ECT towards human application. Using C2C12 mouse myoblasts, our flat-sheet device allowed for long-term continuous antibody delivery in the plasma of subcutaneously implanted mice. We were able to generate antibody-secreting human cells using the immortalized C25 cell It was possible to differentiate these cells into myotubes and adapt them to long-term survival inside the capsules maintained in vitro. However, when implanted in the subcutaneous tissue of immunocompromised mice, myoblasts survived in the ECT device as a thin layer of cells and were not able to secrete recombinant antibodies in sufficient amounts to be considered for therapeutic purpose.

EPFL2019

Parkinson mice show functional and molecular changes in the gut long before motoric disease onset

Hilal Lashuel, Yang Liu

Background: There is increasing evidence that Parkinson's disease (PD) might start in the gut, thus involving and compromising also the enteric nervous system (ENS). At the clinical onset of the disease the majority of dopaminergic neurons in the midbrain is already destroyed, so that the lack of early biomarkers for the disease represents a major challenge for developing timely treatment interventions. Here, we use a transgenic A30P-alpha-synuclein-overexpressing PD mouse model to identify appropriate candidate markers in the gut before hallmark symptoms begin to manifest. Methods: Based on a gait analysis and striatal dopamine levels, we defined 2-month-old A30P mice as pre-symptomatic (psA30P), since they are not showing any motoric impairments of the skeletal neuromuscular system and no reduced dopamine levels, but an intestinal alpha-synuclein pathology. Mice at this particular age were further used to analyze functional and molecular alterations in both, the gastrointestinal tract and the ENS, to identify early pathological changes. We examined the gastrointestinal motility, the molecular composition of the ENS, as well as the expression of regulating miRNAs. Moreover, we applied A30P-alpha-synuclein challenges in vitro to simulate PD in the ENS. Results: A retarded gut motility and early molecular dysregulations were found in the myenteric plexus of psA30P mice. We found that i.e. neurofilament light chain, vesicle-associated membrane protein 2 and calbindin 2, together with the miRNAs that regulate them, are significantly altered in the psA30P, thus representing potential biomarkers for early PD. Many of the dysregulated miRNAs found in the psA30P mice are reported to be changed in PD patients as well, either in blood, cerebrospinal fluid or brain tissue. Interestingly, the in vitro approaches delivered similar changes in the ENS cultures as seen in the transgenic animals, thus confirming the data from the mouse model. Conclusions: These findings provide an interesting and novel approach for the identification of appropriate biomarkers in men.

BMC2021