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.

Prenatal treatment with EGCG enriched green tea extract rescues GAD67 related developmental and cognitive defects in Down syndrome mouse models.

Down syndrome is a common genetic disorder caused by trisomy of chromosome 21. Brain development in affected foetuses might be improved through prenatal treatment. One potential target is DYRK1A, a multifunctional kinase encoded by chromosome 21 that, when overexpressed, alters neuronal excitation-inhibition balance and increases GAD67 interneuron density. We used a green tea extract enriched in EGCG to inhibit DYRK1A function only during gestation of transgenic mice overexpressing Dyrk1a (mBACtgDyrk1a). Adult mice treated prenatally displayed reduced levels of inhibitory markers, restored VGAT1/VGLUT1 balance, and rescued density of GAD67 interneurons. Similar results for gabaergic and glutamatergic markers and interneuron density were obtained in Dp(16)1Yey mice, trisomic for 140 chromosome 21 orthologs; thus, prenatal EGCG exhibits efficacy in a more complex DS model. Finally, cognitive and behaviour testing showed that adult Dp(16)1Yey mice treated prenatally had improved novel object recognition memory but do not show improvement with Y maze paradigm. These findings provide empirical support for a prenatal intervention that targets specific neural circuitries.

Overexpression of the DYRK1A Gene (Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 1A) Induces Alterations of the Serotoninergic and Dopaminergic Processing in Murine Brain Tissues.

Trisomy 21 (T21) or Down syndrome (DS) is the most common genetic disorder associated with intellectual disability and affects around 5 million persons worldwide. Neuroanatomical phenotypes associated with T21 include slight reduction of brain size and weight, abnormalities in several brain areas including spines dysgenesis, dendritic morphogenesis, and early neuroanatomical characteristics of Alzheimer's disease. Monoamine neurotransmitters are involved in dendrites development, functioning of synapses, memory consolidation, and their levels measured in the cerebrospinal fluid, blood, or brain areas that are modified in individuals with T21. DYRK1A is one of the recognized key genes that could explain some of the deficits present in individuals with T21. We investigated by high-performance liquid chromatography with electrochemical detection the contents and processing of monoamines neurotransmitters in four brain areas of female and male transgenic mice for the Dyrk1a gene (mBactgDyrk1a). DYRK1A overexpression induced dramatic deficits in the serotonin contents of the four brain areas tested and major deficits in dopamine and adrenaline contents especially in the hypothalamus. These results suggest that DYRK1A overexpression might be associated with the modification of monoamines content found in individuals with T21 and reinforce the interest to target the level of DYRK1A expression as a therapeutic approach for persons with T21.

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.

Specific age-related molecular alterations in the cerebellum of Down syndrome mouse models.

Down syndrome, or trisomy 21, has been modeled with various trisomic and transgenic mice to help understand the consequences of an altered gene dosage in brain development and function. Though Down syndrome has been associated with premature aging, little is known about the molecular and cellular alterations that target brain function. To help identify alterations at specific ages, we analyzed the cerebellum of Ts1Cje mice, trisomic for 77 HSA21 orthologs, at three ages-young (4 months), middle-age (12 months), and old (17 months)-compared to age-matched controls. Quantification of neuronal and glial markers (n=11) revealed increases in GFAP, with an age effect, and S100B, with age and genotype effects. The genotype effect on S100B with age was unexpected as Ts1Cje has only two copies of the S100b gene. Interestingly, the different increase in GFAP observed between Ts1Cje (trisomic segment includes Pcp4 gene) and controls was magnified in TgPCP4 mice (1 extra copy of the human PCP4 gene) at the same age. S100B increase was not found in the TgPCP4 confirming a difference of regulation with aging for GFAP and S100B and excluding the calcium signaling regulator, Pcp4, as a potential candidate for increase of S100B in the Ts1Cje. To understand these differences, comparison of GFAP and S100B immunostainings at young and middle-age were performed. Immunohistochemical detection of differences in GFAP and S100B localization with aging implicate S100B+ oligodendrocytes as a new phenotypic target in this specific aging process.

Pharmacological correction of excitation/inhibition imbalance in Down syndrome mouse models.

Cognitive impairment in Down syndrome (DS) has been linked to increased synaptic inhibition. The underlying mechanisms remain unknown, but memory deficits are rescued in DS mouse models by drugs targeting GABA receptors. Similarly, administration of epigallocatechin gallate (EGCG)-containing extracts rescues cognitive phenotypes in Ts65Dn mice, potentially through GABA pathway. Some developmental and cognitive alterations have been traced to increased expression of the serine-threonine kinase DYRK1A on Hsa21. To better understand excitation/inhibition balance in DS, we investigated the consequences of long-term (1-month) treatment with EGCG-containing extracts in adult mBACtgDyrk1a mice that overexpress Dyrk1a. Administration of POL60 rescued components of GABAergic and glutamatergic pathways in cortex and hippocampus but not cerebellum. An intermediate dose (60 mg/kg) of decaffeinated green tea extract (MGTE) acted on components of both GABAergic and glutamatergic pathways and rescued behavioral deficits as demonstrated on the alternating paradigm, but did not rescue protein level of GABA-synthesizing GAD67. These results indicate that excessive synaptic inhibition in people with DS may be attributable, in large part, to increased DYRK1A dosage. Thus, controlling the level of active DYRK1A is a clear issue for DS therapy. This study also defines a panel of synaptic markers for further characterization of DS treatments in murine models.

Molecular rescue of DYRK1A overexpression in cystathionine beta synthase-deficient mouse brain by enriched environment combined with voluntary exercise.

Hyperhomocysteinemia resulting from cystathionine beta synthase (CBS) deficiency can produce cognitive dysfunction. We recently found that CBS-deficient mice exhibit increased expression of the serine/threonine kinase dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A) in the brain. When dysregulated, DYRK1A contributes to the neurodegeneration, neuronal death, and loss of function observed in neurodegenerative diseases. However, brain plasticity can be improved by interventions like enriched environment combined with voluntary exercise (EE/VE). The present study sought to assess the effects of EE/VE on molecular mechanisms linked to DYRK1A overexpression in the brain of CBS-deficient mice. EE/VE was applied to 3-month-old female CBS-deficient mice for 1 month. Without intervention, CBS-deficient mice exhibited increased DYRK1A and decreased brain-derived neurotrophic factor (BDNF) levels in the cortex and hippocampus. However, EE/VE rescued these altered DYRK1A and BDNF levels in the hippocampus of CBS-deficient mice. We conclude that exercise combined with enriched environment can restore the altered molecular mechanisms in the brain of CBS-deficient mice.

Excitation/inhibition balance and learning are modified by Dyrk1a gene dosage.

Cognitive deficits in Down syndrome (DS) have been linked to increased synaptic inhibition, leading to an imbalance of excitation/inhibition (E/I). Various mouse models and studies from human brains have implicated an HSA21 gene, the serine/threonine kinase DYRK1A, as a candidate for inducing cognitive dysfunction. Here, consequences of alterations in Dyrk1a dosage were assessed in mouse models with varying copy numbers of Dyrk1a: mBACtgDyrk1a, Ts65Dn and Dp(16)1Yey (with 3 gene copies) and Dyrk1a(+/-) (one functional copy). Molecular (i.e. immunoblotting/immunohistochemistry) and behavioral analyses (e.g., rotarod, Morris water maze, Y-maze) were performed in mBACtgDyrk1a mice. Increased expression of DYRK1A in mBACtgDyrk1a induced molecular alterations in synaptic plasticity pathways, particularly expression changes in GABAergic and glutaminergic related proteins. Similar alterations were observed in models with partial trisomy of MMU16, Ts65Dn and Dp(16)1Yey, and were reversed in the Dyrk1a(+/-) model. Dyrk1a overexpression produced an increased number and signal intensity of GAD67 positive neurons, indicating enhanced inhibition pathways in three different models: mBACtgDyrk1a, hYACtgDyrk1a and Dp(16)1Yey. Functionally, Dyrk1a overexpression protected mice from PTZ-induced seizures related to GABAergic neuron plasticity. Our study shows that DYRK1A overexpression affects pathways involved in synaptogenesis and synaptic plasticity and influences E/I balance toward inhibition. Inhibition of DYRK1A activity offers a therapeutic target for DS, but its inhibition/activation may also be relevant for other psychiatric diseases with E/I balance alterations.

Prefrontal deficits in a murine model overexpressing the down syndrome candidate gene dyrk1a.

The gene Dyrk1a is the mammalian ortholog of Drosophila minibrain. Dyrk1a localizes in the Down syndrome (DS) critical region of chromosome 21q22.2 and is a major candidate for the behavioral and neuronal abnormalities associated with DS. PFC malfunctions are a common denominator in several neuropsychiatric diseases, including DS, but the contribution of DYRK1A in PFC dysfunctions, in particular the synaptic basis for impairments of executive functions reported in DS patients, remains obscure. We quantified synaptic plasticity, biochemical synaptic markers, and dendritic morphology of deep layer pyramidal PFC neurons in adult mBACtgDyrk1a transgenic mice that overexpress Dyrk1a under the control of its own regulatory sequences. We found that overexpression of Dyrk1a largely increased the number of spines on oblique dendrites of pyramidal neurons, as evidenced by augmented spine density, higher PSD95 protein levels, and larger miniature EPSCs. The dendritic alterations were associated with anomalous NMDAR-mediated long-term potentiation and accompanied by a marked reduction in the pCaMKII/CaMKII ratio in mBACtgDyrk1a mice. Retrograde endocannabinoid-mediated long-term depression (eCB-LTD) was ablated in mBACtgDyrk1a mice. Administration of green tea extracts containing epigallocatechin 3-gallate, a potent DYRK1A inhibitor, to adult mBACtgDyrk1a mice normalized long-term potentiation and spine anomalies but not eCB-LTD. However, inhibition of the eCB deactivating enzyme monoacylglycerol lipase normalized eCB-LTD in mBACtgDyrk1a mice. These data shed light on previously undisclosed participation of DYRK1A in adult PFC dendritic structures and synaptic plasticity. Furthermore, they suggest its involvement in DS-related endophenotypes and identify new potential therapeutic strategies.

DYRK1A: a master regulatory protein controlling brain growth.

Copy number variation in a small region of chromosome 21 containing DYRK1A produces morphological and cognitive alterations in human. In mouse models, haploinsufficiency results in microcephaly, and a human DYRK1A gain-of-function model (three alleles) exhibits increased brain volume. To investigate these developmental aspects, we used a murine BAC clone containing the entire gene to construct an overexpression model driven by endogenous regulatory sequences. We compared this new model to two other mouse models with three copies of Dyrk1a, YACtgDyrk1a and Ts65Dn, as well as the loss-of-function model with one copy (Dyrk1a(+/-)). Growth, viability, brain weight, and brain volume depended strongly upon gene copy number. Brain region-specific variations observed in gain-of-function models mirror their counterparts in the loss-of-function model. Some variations, such as increased volume of the superior colliculus and ventricles, were observed in both the BAC transgenic and Ts65Dn mice. Using unbiased stereology we found that, in the cortex, neuron density is inversely related to Dyrk1a copy number but, in thalamic nuclei, neuron density is directly related to copy number. In addition, six genes involved either in cell division (Ccnd1 and pAkt) or in neuronal machinery (Gap43, Map2, Syp, Snap25) were regulated by Dyrk1a throughout development, from birth to adult. These results imply that Dyrk1a expression alters different cellular processes during brain development. Dyrk1a, then, has two roles in the development process: shaping the brain and controlling the structure of neuronal components.