Biological Diagnosis of Alzheimer's Disease Based on Amyloid Status: An Illustration of Confirmation Bias in Medical Research?

Alzheimer's disease (AD) was first characterized by Dr. Alois Alzheimer in 1906 by studying a demented patient and discovering cerebral amyloid plaques and neurofibrillary tangles. Subsequent research highlighted the roles of Aß peptides and tau proteins, which are the primary constituents of these lesions, which led to the amyloid cascade hypothesis. Technological advances, such as PET scans using Florbetapir, have made it possible to visualize amyloid plaques in living patients, thus improving AD's risk assessment. The National Institute on Aging and the Alzheimer's Association introduced biological diagnostic criteria in 2011, which underlined the amyloid deposits diagnostic value. However, potential confirmation bias may have led researchers to over-rely on amyloid markers independent of AD's symptoms, despite evidence of their limited specificity. This review provides a critical examination of the current research paradigm in AD, including, in particular, the predominant focus on amyloid and tau species in diagnostics. We discuss the potential multifaceted consequences of this approach and propose strategies to mitigate its overemphasis in the development of new biomarkers. Furthermore, our study presents comprehensive guidelines aimed at enhancing the creation of biomarkers for accurately predicting AD dementia onset. These innovations are crucial for refining patient selection processes in clinical trial enrollment and for the optimization of therapeutic strategies. Overcoming confirmation bias is essential to advance the diagnosis and treatment of AD and to move towards precision medicine by incorporating a more nuanced understanding of amyloid biomarkers.

Real-Time Monitoring of Exosome Enveloped-AAV Spreading by Endomicroscopy Approach: A New Tool for Gene Delivery in the Brain.

Exosomes represent a strategy for optimizing the adeno-associated virus (AAV) toward the development of novel therapeutic options for neurodegenerative disorders. However, in vivo spreading of exosomes and AAVs after intracerebral administration is poorly understood. This study provides an assessment and comparison of the spreading into the brain of exosome-enveloped AAVs (exo-AAVs) or unassociated AAVs (std-AAVs) through in vivo optical imaging techniques like probe-based confocal laser endomicroscopy (pCLE) and ex vivo fluorescence microscopy. The std-AAV serotypes (AAV6 and AAV9) encoding the GFP were enveloped in exosomes and injected into the ipsilateral hippocampus. At 3 months post-injection, pCLE detected enhanced GFP expression of both exo-AAV serotypes in contralateral hemispheres compared to std-AAVs. Although sparse GFP-positive astrocytes were observed using exo-AAVs, our results show that the enhancement of the transgene expression resulting from exo-AAVs was largely restricted to neurons and oligodendrocytes. Our results suggest (1) the possibility of combining gene therapy with an endoscopic approach to enable tracking of exo-AAV spread, and (2) exo-AAVs allow for widespread, long-term gene expression in the CNS, supporting the use of exo-AAVs as an efficient gene delivery tool.

Inhibition of DYRK1A proteolysis modifies its kinase specificity and rescues Alzheimer phenotype in APP/PS1 mice.

Recent evidences suggest the involvement of DYRK1A (dual specificity tyrosine phosphorylation-regulated kinase 1 A) in Alzheimer's disease (AD). Here we showed that DYRK1A undergoes a proteolytic processing in AD patients hippocampus without consequences on its kinase activity. Resulting truncated forms accumulate in astrocytes and exhibit increased affinity towards STAT3ɑ, a regulator of inflammatory process. These findings were confirmed in APP/PS1 mice, an amyloid model of AD, suggesting that this DYRK1A cleavage is a consequence of the amyloid pathology. We identified in vitro the Leucettine L41 as a compound able to prevent DYRK1A proteolysis in both human and mouse protein extracts. We then showed that intraperitoneal injections of L41 in aged APP/PS1 mice inhibit STAT3ɑ phosphorylation and reduce pro-inflammatory cytokines levels (IL1- ß, TNF-ɑ and IL-12) associated to an increased microglial recruitment around amyloid plaques and decreased amyloid-ß plaque burden. Importantly, L41 treatment improved synaptic plasticity and rescued memory functions in APP/PS1 mice. Collectively, our results suggest that DYRK1A may contribute to AD pathology through its proteolytic process, reducing its kinase specificity. Further evaluation of inhibitors of DYRK1A truncation promises a new therapeutic approach for AD.

Transient increase in sAPPα secretion in response to Aß1-42 oligomers: an attempt of neuronal self-defense?

Amyloid precursor protein (APP), a key molecule of Alzheimer disease, is metabolized in 2 antagonist pathways generating the soluble APP alpha (sAPPα) having neuroprotective properties and the beta amyloid (Aß) peptide at the origin of neurotoxic oligomers, particularly Aß1-42. Whether extracellular Aß1-42 oligomers modulate the formation and secretion of sAPPα is not known. We report here that the addition of Aß1-42 oligomers to primary cortical neurons induced a transient increase in α-secretase activity and secreted sAPPα 6-9 hours later. Preventing the generation of sAPPα by using small interfering RNAs (siRNAs) for the α-secretases ADAM10 and ADAM17 or for APP led to increased Aß1-42 oligomer-induced cell death after 24 hours. Neuronal injuries due to oxidative stress or growth factor deprivation also generated sAPPα 7 hours later. Finally, acute injection of Aß1-42 oligomers into wild-type mouse hippocampi induced transient secretion of sAPPα 48-72 hours later. Altogether, these data suggest that neurons respond to stress by generating sAPPα for their survival. These data must be taken into account when interpreting sAPPα levels as a biomarker in neurological disorders.

ßAPP Processing Drives Gradual Tau Pathology in an Age-Dependent Amyloid Rat Model of Alzheimer's Disease.

The treatment of Alzheimer's disease (AD) remains challenging and requires a better in depth understanding of AD progression. Particularly, the link between amyloid protein precursor (APP) processing and Tau pathology development remains poorly understood. Growing evidences suggest that APP processing and amyloid-ß (Aß) release are upstream of Tau pathology but the lack of animal models mimicking the slow progression of human AD raised questions around this mechanism. Here, we described that an AD-like ßAPP processing in adults wild-type rats, yielding to human APP, ßCTF and Aß levels similar to those observed in AD patients, is sufficient to trigger gradual Tauopathy. The Tau hyperphosphorylation begins several months before the formation of both amyloid plaques and tangle-like aggregates in aged rats and without associated inflammation. Based on a longitudinal characterization over 30 months, we showed that extrasynaptic and emotional impairments appear before long-term potentiation deficits and memory decline and so before Aß and Tau aggregations. These compelling data allowed us to (1) experimentally confirm the causal relationship between ßAPP processing and Tau pathology in vivo and without Tau transgene overexpression, (2) support the amyloidogenic cascade and (3) propose a 4-step hypothesis of prodromal AD progression.

Interleukin-2 improves amyloid pathology, synaptic failure and memory in Alzheimer's disease mice.

Interleukin-2 (IL-2)-deficient mice have cytoarchitectural hippocampal modifications and impaired learning and memory ability reminiscent of Alzheimer's disease. IL-2 stimulates regulatory T cells whose role is to control inflammation. As neuroinflammation contributes to neurodegeneration, we investigated IL-2 in Alzheimer's disease. Therefore, we investigated IL-2 levels in hippocampal biopsies of patients with Alzheimer's disease relative to age-matched control individuals. We then treated APP/PS1ΔE9 mice having established Alzheimer's disease with IL-2 for 5 months using single administration of an AAV-IL-2 vector. We first found decreased IL-2 levels in hippocampal biopsies of patients with Alzheimer's disease. In mice, IL-2-induced systemic and brain regulatory T cells expansion and activation. In the hippocampus, IL-2 induced astrocytic activation and recruitment of astrocytes around amyloid plaques, decreased amyloid-ß42/40 ratio and amyloid plaque load, improved synaptic plasticity and significantly rescued spine density. Of note, this tissue remodelling was associated with recovery of memory deficits, as assessed in the Morris water maze task. Altogether, our data strongly suggest that IL-2 can alleviate Alzheimer's disease hallmarks in APP/PS1ΔE9 mice with established pathology. Therefore, this should prompt the investigation of low-dose IL-2 in Alzheimer's disease and other neuroinflammatory/neurodegenerative disorders.

Alzheimer's disease-like APP processing in wild-type mice identifies synaptic defects as initial steps of disease progression.

BACKGROUND: Alzheimer's disease (AD) is the most frequent form of dementia in the elderly and no effective treatment is currently available. The mechanisms triggering AD onset and progression are still imperfectly dissected. We aimed at deciphering the modifications occurring in vivo during the very early stages of AD, before the development of amyloid deposits, neurofibrillary tangles, neuronal death and inflammation. Most current AD models based on Amyloid Precursor Protein (APP) overproduction beginning from in utero, to rapidly reproduce the histological and behavioral features of the disease within a few months, are not appropriate to study the early steps of AD development. As a means to mimic in vivo amyloid APP processing closer to the human situation in AD, we used an adeno-associated virus (AAV)-based transfer of human mutant APP and Presenilin 1 (PS1) genes to the hippocampi of two-month-old C57Bl/6 J mice to express human APP, without significant overexpression and to specifically induce its amyloid processing. RESULTS: The human APP, ßCTF and Aß42/40 ratio were similar to those in hippocampal tissues from AD patients. Three months after injection the murine Tau protein was hyperphosphorylated and rapid synaptic failure occurred characterized by decreased levels of both PSD-95 and metabolites related to neuromodulation, on proton magnetic resonance spectroscopy ((1)H-MRS). Astrocytic GLT-1 transporter levels were lower and the tonic glutamatergic current was stronger on electrophysiological recordings of CA1 hippocampal region, revealing the overstimulation of extrasynaptic N-methyl D-aspartate receptor (NMDAR) which precedes the loss of long-term potentiation (LTP). These modifications were associated with early behavioral impairments in the Open-field, Y-maze and Morris Mater Maze tasks. CONCLUSIONS: Altogether, this demonstrates that an AD-like APP processing, yielding to levels of APP, ßCTF and Aß42/Aß40 ratio similar to those observed in AD patients, are sufficient to rapidly trigger early steps of the amyloidogenic and Tau pathways in vivo. With this strategy, we identified a sequence of early events likely to account for disease onset and described a model that may facilitate efforts to decipher the factors triggering AD and to evaluate early neuroprotective strategies.

Viral gene transfer of APPsα rescues synaptic failure in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is characterized by synaptic failure, dendritic and axonal atrophy, neuronal death and progressive loss of cognitive functions. It is commonly assumed that these deficits arise due to ß-amyloid accumulation and plaque deposition. However, increasing evidence indicates that loss of physiological APP functions mediated predominantly by neurotrophic APPsα produced in the non-amyloidogenic α-secretase pathway may contribute to AD pathogenesis. Upregulation of APPsα production via induction of α-secretase might, however, be problematic as this may also affect substrates implicated in tumorigenesis. Here, we used a gene therapy approach to directly overexpress APPsα in the brain using AAV-mediated gene transfer and explored its potential to rescue structural, electrophysiological and behavioral deficits in APP/PS1∆E9 AD model mice. Sustained APPsα overexpression in aged mice with already preexisting pathology and amyloidosis restored synaptic plasticity and partially rescued spine density deficits. Importantly, AAV-APPsα treatment also resulted in a functional rescue of spatial reference memory in the Morris water maze. Moreover, we demonstrate a significant reduction of soluble Aß species and plaque load. In addition, APPsα induced the recruitment of microglia with a ramified morphology into the vicinity of plaques and upregulated IDE and TREM2 expression suggesting enhanced plaque clearance. Collectively, these data indicate that APPsα can mitigate synaptic and cognitive deficits, despite established pathology. Increasing APPsα may therefore be of therapeutic relevance for AD.

Cholesterol 24-hydroxylase defect is implicated in memory impairments associated with Alzheimer-like Tau pathology.

Alzheimer's disease (AD) is characterized by both amyloid and Tau pathologies. The amyloid component and altered cholesterol metabolism are closely linked, but the relationship between Tau pathology and cholesterol is currently unclear. Brain cholesterol is synthesized in situ and cannot cross the blood-brain barrier: to be exported from the central nervous system into the blood circuit, excess cholesterol must be converted to 24S-hydroxycholesterol by the cholesterol 24-hydroxylase encoded by the CYP46A1 gene. In AD patients, the concentration of 24S-hydroxycholesterol in the plasma and the cerebrospinal fluid are lower than in healthy controls. The THY-Tau22 mouse is a model of AD-like Tau pathology without amyloid pathology. We used this model to investigate the potential association between Tau pathology and CYP46A1 modulation. The amounts of CYP46A1 and 24S-hydroxycholesterol in the hippocampus were lower in THY-Tau22 than control mice. We used an adeno-associated virus (AAV) gene transfer strategy to increase CYP46A1 expression in order to investigate the consequences on THY-Tau22 mouse phenotype. Injection of the AAV-CYP46A1 vector into the hippocampus of THY-Tau22 mice led to CYP46A1 and 24S-hydroxycholesterol content normalization. The cognitive deficits, impaired long-term depression and spine defects that characterize the THY-Tau22 model were completely rescued, whereas Tau hyperphosphorylation and associated gliosis were unaffected. These results argue for a causal link between CYP46A1 protein content and memory impairments that result from Tau pathology. Therefore, CYP46A1 may be a relevant therapeutic target for Tauopathies and especially for AD.

CYP46A1 inhibition, brain cholesterol accumulation and neurodegeneration pave the way for Alzheimer's disease.

Abnormalities in neuronal cholesterol homeostasis have been suspected or observed in several neurodegenerative disorders including Alzheimer's disease, Parkinson's disease and Huntington's disease. However, it has not been demonstrated whether an increased abundance of cholesterol in neurons in vivo contributes to neurodegeneration. To address this issue, we used RNA interference methodology to inhibit the expression of cholesterol 24-hydroxylase, encoded by the Cyp46a1 gene, in the hippocampus of normal mice. Cholesterol 24-hydroxylase controls cholesterol efflux from the brain and thereby plays a major role in regulating brain cholesterol homeostasis. We used an adeno-associated virus vector encoding short hairpin RNA directed against the mouse Cyp46a1 mRNA to decrease the expression of the Cyp46a1 gene in hippocampal neurons of normal mice. This increased the cholesterol concentration in neurons, followed by cognitive deficits and hippocampal atrophy due to apoptotic neuronal death. Prior to neuronal death, the recruitment of the amyloid protein precursor to lipid rafts was enhanced leading to the production of ß-C-terminal fragment and amyloid-ß peptides. Abnormal phosphorylation of tau and endoplasmic reticulum stress were also observed. In the APP23 mouse model of Alzheimer's disease, the abundance of amyloid-ß peptides increased following inhibition of Cyp46a1 expression, and neuronal death was more widespread than in normal mice. Altogether, these results suggest that increased amounts of neuronal cholesterol within the brain may contribute to inducing and/or aggravating Alzheimer's disease.

Reducing GABAergic inhibition restores cognitive functions in a mouse model of Down syndrome.

Alterations in excitatory-inhibitory balance occur in Down syndrome and could be responsible for cognitive deficits observed through the life of all individuals carrying an extra copy of chromosome 21. Excess of inhibition in the adult could produce synaptic plasticity deficits that may be a primary mechanism contributing to learning and memory impairments. In this study we discuss pharmacological treatments that could potentially alleviate neuronal inhibition and have been tested in a mouse model of Down syndrome. γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mature central nervous system that binds to GABA-benzodiazepine receptors, opens a chloride channel and reduces neuronal excitability. These receptors have been extensively studied as targets for treatment of epilepsy, anxiety, sleep, cognitive disorders and the induction of sedation. Molecules that are either antagonists or inverse agonists of the GABA-benzodiazepine receptors are able to reduce inhibitory GABAergic transmission. However modulating the excitatory-inhibitory balance towards increase of cognition without inducing seizures remains difficult particularly when using GABA antagonists. In this study we review data from the literature obtained using inverse agonists selective for the α5-subunit containing receptor. Such inverse agonists, initially developed as cognitive enhancers for treatment of memory impairments, proved to be very efficient in reversing learning and memory deficits in a Down syndrome mouse model after acute treatment.

PTD-XIAP protects against cerebral ischemia by anti-apoptotic and transcriptional regulatory mechanisms.

Caspases play a major role in the infarction process that follows occlusion of cerebral arteries and are important targets for stroke therapy. We have generated three fusion proteins that link various domains of the X chromosome-linked inhibitor of apoptosis (XIAP), a potent caspase inhibitor, to the protein transduction domain (PTD) of HIV-1/Tat, and have tested their efficacy after distal occlusion of the middle cerebral artery (dMCAO) in mice. PTD-XIAP failed to accumulate in brain structures after intravenous (iv) delivery, but properly transduced cortical cells when applied topically. Shorter constructs efficiently targeted the lesion after iv delivery. All proteins retained their caspase inhibitory activity and significantly reduced infarct volumes. PTD-XIAP reversed long-term impairments in the water maze test. Sequential activation of transcription factors was observed, suggesting that the effects of XIAP are mediated by both direct inhibition of apoptotic mechanisms and secondary regulation of transcription factors involved in neuronal survival.

Activation of proinflammatory caspases by cathepsin B in focal cerebral ischemia.

Cathepsins and caspases are two families of proteases that play pivotal roles in ischemic cell death. This study investigated the existence of a cross-talk between cathepsin B and proinflammatory caspases in stroke-induced cell death, as recently suggested by in vitro data. Cortical ischemic damage was induced in mice by distal and permanent occlusion of the middle cerebral artery. Cytoplasmic activation of cathepsin B was observed from the early stages of infarction, and displayed an activation pattern parallel to the activation pattern of caspase-1 and -11. Immunohistochemistry revealed the colocalization of cathepsin B with each caspase in cells of the infarct core. The apical position of cathepsin B in both caspase-activation cascades was confirmed by pretreatment of the animals with the cathepsin B inhibitor CA-074, which also potently protected cortical structures from ischemic damage, indicating involvement of the proteases in the lesion process. The results show that cathepsin B release is an early event following occlusion of cerebral arteries, which eventually triggers the activation of proinflammatory caspases in the absence of reperfusion. This new pathway may play a critical role in brain infarction by promoting inflammatory responses, and/or by amplifying the apoptotic process.

The mechanisms of cell death in focal cerebral ischemia highlight neuroprotective perspectives by anti-caspase therapy.

A number of studies have validated the importance of caspase activation in ischemia-induced brain damage. Caspases participate in both the initiation and execution phases of apoptosis, and play a central role in neuronal death after global cerebral ischemia. In focal ischemia, apoptosis occurs in the penumbra during the secondary phase of expansion of the lesion. However, ultrastructural and biochemical analysis have also shown signs of apoptosis in the initial lesion, or infarct core, which is traditionally considered necrotic. Specific caspase pathways are activated in the core and in the penumbra, and participate in both cytoplasmic and nuclear apoptotic events, notwithstanding their initial classification as activator or initiator caspases. This confirms previous suggestions that caspase inhibition holds tremendous neuroprotective potential in stroke and other apoptosis-related degenerative diseases. Consequently, two new approaches, aimed at treating stroke-induced brain damage by anti-apoptotic molecules, are being developed in academic and industrial laboratories. These are based, respectively, on the use of small peptide sequences corresponding to the preferred cleavage site of a caspase, and on genomic constructions derived from the fusion of endogenous anti-caspase molecules with a protein transduction domain from the human immunodeficiency virus-1. Fusion proteins containing endogenous caspases inhibitors efficiently counteract apoptosis in vitro. In in vivo models of focal cerebral ischemia, fusion proteins successfully cross the blood brain barrier and protect cells from ischemic death. This new approach by protein therapy could prove to be an interesting alternative for the reduction of the dramatic consequences of stroke, provided that the long-term efficiency of this protection in terms of functional recovery is demonstrated.