Multimodal phenotyping of synaptic damage in Alzheimer’s disease : translational perspective with focus on quantitative EEG

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia. Accumulation of AD-associated pathology in the brain may begin a decade or more before the appearance of the first symptoms of the disease. The pathological-clinical “continuum of AD” therefore encompasses time between the initial neuropathological changes and symptoms of advanced disease. Besides cognitively healthy individuals at risk, it includes subjects with subjective cognitive decline (SCD), mild cognitive impairment (MCI) and eventually dementia when the severity of cognitive impairment affects patients’ ability to carry out everyday activities. Timely detection of the disease would therefore recognize patients that are at risk for future cognitive deterioration and provide time window for the prevention and novel therapeutical interventions. Accumulating evidence suggests that degeneration and dysfunction of brain neuronal connections, i.e. synapses, is one of the earliest and best proxies of cognitive deficits in patients along AD continuum. Human electroencephalography (EEG) is a non-invasive and widely available diagnostic method that records real-time large-scale synaptic activity. The commonly used method in research settings is quantitative EEG (qEEG) analysis that provides objective information on EEG recorded at the level of the scalp. Quantitative EEG analysis unravels complex EEG signal and adds relevant information on its spectral components (frequency domain), temporal dynamics (time domain) and topographic estimates (space domain) of brain cortical activity. The general aim of the present thesis was to characterize different aspects of synaptic degeneration in AD, with the focus on qEEG and its relationship to both conventional and novel synaptic markers. In study I, global qEEG measures of power and synchronization were found to correlate with conventional cerebrospinal fluid (CSF) biomarkers of Aβ and tau pathology in patients diagnosed with SCD, MCI and AD, linking the markers of AD pathology to the generalized EEG slowing and reduced brain connectivity in fast frequency bands. In study II, qEEG analysis in the time domain (EEG microstates) revealed alterations in the organization and dynamics of large-scale brain networks in memory clinic patients compared to healthy elderly controls. In study III, topographical qEEG analysis of brain functional connectivity was associated with regionspecific cortical glucose hypometabolism ([18F]Fluorodeoxyglucose positron-emission tomography) in MCI and AD patients. Study IV provided evidence that qEEG measures of global power and synchronization correlate with CSF levels of synaptic marker neurogranin, both modalities being in combination independent predictors of progression to AD dementia in MCI patients. Study V and associated preliminary study introduced in the thesis assessed the translational potential of CSF neurogranin and qEEG as well as their direct relationship to AD neuropathology in App knock-in mouse models of AD. In study V, changes in CSF neurogranin levels and their relationship to conventional CSF markers in App knock-in mice corresponded to the pattern observed in clinical AD cohorts. These findings highlighted the potential use of mouse CSF biomarkers as well as App knock-in mouse models for translational investigation of synaptic dysfunction due to AD. In general, the results of the thesis invite for further clinical validation of multimodal synaptic markers in the context of early AD diagnosis, prognosis, and treatment monitoring in individual patients

A qualitative impairment in face perception in Alzheimer’s disease : evidence from a reduced face inversion effect

Prevalent face recognition difficulties in Alzheimer’s disease (AD) have typically been attributed to the underlying episodic and semantic memory impairment. The aim of the current study was to determine if AD patients are also impaired at the perceptual level for faces, more specifically at extracting a visual representation of an individual face. To address this question, we investigated the matching of simultaneously presented individual faces and of other nonface familiar shapes (cars), at both upright and inverted orientation, in a group of mild AD patients and in a group of healthy older controls matched for age and education. AD patients showed a reduced inversion effect (i.e., larger performance for upright than inverted stimuli) for faces, but not for cars, both in terms of error rates and response times. While healthy participants showed a much larger decrease in performance for faces than for cars with inversion, the inversion effect did not differ significantly for faces and cars in AD. This abnormal inversion effect for faces was observed in a large subset of individual patients with AD. These results suggest that AD patients have deficits in higher-level visual processes, more specifically at perceiving individual faces, a function that relies on holistic representations specific to upright face stimuli. These deficits, combined with their memory impairment, may contribute to the difficulties in recognizing familiar people that are often reported in patients suffering from the disease and by their caregivers

Using fMRI to investigate speech-stream segregation and auditory attention in healthy adults and patients with memory complaints

Poor memory for recent conversations is the commonest presenting symptom in patients attending a cognitive neurology clinic. They also frequently have greater difficulty following and remembering conversations in the presence of background noise and/or unattended speech. While the ability to participate in and recall conversations depends on several cognitive functions (language-processing, attention, episodic and working memory), without the ability to perform auditory scene analysis, and more specifically speech-stream segregation, recall of verbal information will be impaired as a consequence of poor initial registration, over and above impaired encoding and subsequent retrieval. This thesis investigated auditory attention and speech-stream segregation in healthy participants (‘controls’) and patients presenting with ‘poor memory’, particularly a complaint of difficulty remembering recent verbal information. Although this resulted in the recruitment of many patients with possible or probable Alzheimer’s disease, it also included patients with mild cognitive impairment (MCI) of uncertain aetiology and a few with depression. Functional MRI data revealed brain activity involved in attention, working memory and speech-stream segregation as participants attended to a speaker in the absence and presence of background speech. The study on controls demonstrated that the right anterior insula, adjacent frontal operculum, left planum temporale and precuneus were more active when the attended speaker was partially masked by unattended speech. Analyses also revealed a central role for a right hemisphere system for successful attentive listening, a system that was not modulated by administration of a central cholinesterase inhibitor. Therefore, this study identified non-auditory higher-order regions in speech-stream segregation, and the demands on a right hemisphere system during attentive listening. Administration of a central cholinesterase inhibitor did not identify any benefit in the present patient group. However, my research has identified systems that might be therapeutic targets when attempting to modulate auditory attention and speech-stream segregation in patients with neurodegenerative disease.Open Acces

AGE- AND SEX-DEPENDENT ALTERATIONS IN PRIMARY SOMATOSENSORY NEURONAL CALCIUM NETWORK DYNAMICS DURING LOCOMOTION

Over the past 30 years, the calcium (Ca2+) hypothesis of brain aging has provided clear evidence that hippocampal neuronal Ca2+ dysregulation is a key biomarker of aging. Indeed, age-dependent Ca2+-mediated changes in intrinsic excitability, synaptic plasticity, and activity have helped identify some of the mechanisms engaged in memory and cognitive decline. However, much of this work has been done at the single-cell level, mostly in slice preparations, and in restricted structures of the brain. Recently, our lab identified age- and Ca2+-related neuronal network dysregulation in the cortex of the anesthetized animal. Still, investigations in the awake animal are needed to test the generalizability of the Ca2+ hypothesis of brain aging and dementia. Here, we used in vigilo two-photon (2P) imaging in ambulating mice, to image GCaMP8f in the primary somatosensory cortex (S1), during ambulation and at rest. In order to investigate aging- and sex- related changes in the neuronal Ca2+ network, a continuous wavelet transform (CWT) analysis was developed (MATLAB) to extract measures of network communication while also addressing pair-wise correlations at single-cell resolution. Following imaging, gait behavior was characterized to test for changes in locomotor stability. During ambulation and compared to rest, in both young (4 months) and aged mice (22 months), an increase in connectivity and synchronicity was noted. An age-dependent increase in network synchronicity was seen in ambulating aged males only. Additionally, females displayed a greater number of active neurons, area-under-curve, and neuronal activity compared to males, particularly during ambulation. These results suggest S1 Ca2+ dynamics and network synchronicity are likely contributors of locomotor stability. We believe this work raises awareness of central elements at play in S1 where neuronal Ca2+ network dysregulation is seen with aging, perhaps highlighting potential therapeutic targets that may help offset age-dependent increases in falls

Cholinergic Mechanisms Regulating Cognitive Function and RNA Metabolism

Acetylcholine (ACh) is one of the main neuromodulators in the mammalian central nervous system (CNS). This chemical messenger has been implicated in the underlying physiology of many distinct cognitive functions. However, the exact role that ACh plays in regulating information processing in the brain is still not fully understood. The vesicular acetylcholine transporter (VAChT) is required for the storage of ACh into synaptic vesicles, and therefore it presents a means to modulate release. Diminished VAChT levels cause a decrease in cholinergic tone, whereas increased VAChT expression has been shown to augment ACh release. Previously published data have shown that elimination of VAChT in the basal forebrain in genetically-modified mice impairs learning and memory. For our studies we have used different mouse lines in which the expression of the VAChT gene is changed, both increased and decreased. We are therefore able to study the consequences of altered cholinergic tone in vivo. Our hypothesis is that changes in cholinergic tone produce specific molecular signatures in target brain areas that underlie alterations in cognitive function. Our studies aimed to elucidate the behavioural and molecular consequences of cholinergic dysfunction. Behavioral testing included classical learning and memory tests as well as sophisticated tasks using novel touch screens chambers to measure attention, learning and memory as well as cognitive flexibility. At the molecular level, the goal was to examine how long-term changes in cholinergic tone impact mechanisms regulating synaptic plasticity and neuronal health. Finally, by aging mouse models of cholinergic dysfunction we were able to elucidate the role that cholinergic tone plays in the classical pathological hallmarks of neurodegenerative disorders. Ultimately, by establishing the molecular signature of increased and decreased cholinergic tone in targeted brain regions (cortex and hippocampus) it may become possible to find novel targets for therapeutic interventions to improve cognitive deficits due to altered cholinergic tone

Hippocampus

The hippocampus is a bicortical structure with extensive fiber connections with multiple brain regions. It is involved in several functions, such as learning, memory, attention, emotion, and more. This book covers various aspects of the hippocampus including cytoarchitecture, functions, diseases, and treatment. It highlights the most advanced findings in research on the hippocampus. It discusses circuits, pattern formation process of grid cells, and zinc dynamics of the hippocampus. The book also addresses the tau pathology and circRNAs related to Alzheimer’s disease and potential treatment strategies. It is a useful resource for general readers, students, and researchers

Hippocampal neural disinhibition causes attentional and memory deficits

Subconvulsive hippocampal neural disinhibition, that is reduced GABAergic inhibition, has been implicated in neuropsychiatric disorders characterized by attentional and memory deficits, including schizophrenia and age-related cognitive decline. Considering that neural disinhibition may disrupt both intra-hippocampal processing and processing in hippocampal projection sites, we hypothesized that hippocampal disinhibition disrupts hippocampus-dependent memory performance and, based on strong hippocampo-prefrontal connectivity, also prefrontal-dependent attention. In support of this hypothesis, we report that acute hippocampal disinhibition by microinfusion of the GABA-A receptor antagonist picrotoxin in rats impaired hippocampus-dependent everyday-type rapid place learning performance on the watermaze delayed-matching-to-place test and prefrontal-dependent attentional performance on the 5-choice-serial-reaction-time test, which does not normally require the hippocampus. For comparison, we also examined psychosis-related sensorimotor effects, using startle/prepulse inhibition (PPI) and locomotor testing. Hippocampal picrotoxin moderately increased locomotion and slightly reduced startle reactivity, without affecting PPI. In vivo electrophysiological recordings in the vicinity of the infusion site showed that picrotoxin mainly enhanced burst firing of hippocampal neurons. In conclusion, hippocampal neural disinhibition disrupts hippocampus-dependent memory performance and also manifests through deficits in not normally hippocampus-dependent attentional performance. These behavioral deficits may reflect a disrupted control of burst firing, which may disrupt hippocampal processing and cause aberrant drive to hippocampal projection sites

Redes metabólicas no envelhecimento e doenças relacionadas com o envelhecimento

Aging is a natural physiological process, but its specific causes are not entirely understood at the molecular level. During aging, the levels of the redox cofactor Nicotinamide Adenine Dinucleotide (NAD) decrease. This molecule is essential for energy production by the cell and is also a substrate to a range of enzymes that regulate gene expression and cell survival. To gain insight into the metabolic networks of age-related disorders, we took a combined bioinformatics and molecular approach. We used text-mining methods to extract protein interaction data from 1500 PubMed abstracts containing keywords related to proteostasis, aging and age-related diseases. Protein networks were obtained with Cytoscape and were submitted to parameter-based analysis. An enrichment analysis using the cytoscape plug-in ClueGo was followed. Parameter analysis revealed APP as the most central and influential protein in the network and enrichment analysis depicted a predominance of terms related to Immune system along with Cancer and Cell Cycle regulation. As a cellular model, we have used a NAD metabolism inhibitor in SH-SY5Y neuroblastoma cells to mimic the NAD decline during aging. At different time points (8h, 24h, and 48h) we measured cell viability along with the expression levels of NAMPT and NAPRT, the rate-limiting enzymes from the NAD biosynthetic nicotinamide salvage pathway and the Preiss handler pathway respectively. Our results show a 50% decrease in cell viability at 48h along with a decrease in NAPRT protein expression. No alterations in NAMPT protein levels were recorded in any measured time point. It is possible that other NAD biosynthesis pathways are activated, so further studies intended to elucidate question are required.O envelhecimento é um processo fisiológico natural, contudo as suas causas específicas não são totalmente compreendidas ao nível molecular. Durante o envelhecimento, os níveis do cofator redox Nicotinamida Adenina Dinucleotídeo (NAD+) diminuem. Esta molécula é essencial para a produção de energia por parte da célula e também é um substrato para uma variedade de enzimas que regulam a expressão genética e a sobrevivência celular. Para obter informações sobre as redes metabólicas de doenças relacionadas com o envelhecimento, adotamos uma combinação de abordagens bioinformática e molecular. Utilizamos métodos de extração de texto para extrair dados de interação proteica de 1500 resumos de artigos da PubMed contendo palavras-chave relacionadas com proteostase, envelhecimento e doenças relacionadas com o envelhecimento. As redes de proteínas foram obtidas com o Cytoscape e submetidas a uma análise baseada em parâmetros. Seguiu-se uma análise de enriquecimento usando o ClueGo um plug-in do Cytoscape. A análise de parâmetros revelou APP como a proteína mais central e influente na rede e a análise de enriquecimento retratou uma predominância de termos relacionados com o sistema imunológico, juntamente com a regulação do ciclo celular. Como modelo celular, usamos um inibidor do metabolismo do NAD+ em células de neuroblastoma SH-SY5Y para imitar o declínio do NAD+ durante o envelhecimento. Em diferentes momentos (8h, 24h, 48h e 72h), medimos a viabilidade celular juntamente com os níveis de expressão de NAMPT e NAPRT, as enzimas limitadoras de taxa das vias Salvage de Nicotinamida e Preiss-Handler, respetivamente, ambas vias de produtoras de NAD+. Os Nossos resultados mostram uma diminuição de 50% na viabilidade celular às 48h, juntamente com uma diminuição na expressão proteica de NAPRT. Não foram registadas alterações nos níveis de proteína NAMPT em nenhum momento. É possível que outras vias de biossíntese do NAD+ estejam ativas, de maneira que outros estudos com o objetivo de elucidar esta questão são necessários.Mestrado em Biomedicina Molecula

Integrative Analysis to Investigate Complex Interaction in Alzheimer’s Disease

Alzheimer’s disease (AD) is a neurodegenerative disorder featuring progressive cognitive and functional deficits. Pathologically, AD is characterized by tau and amyloid β protein deposition in the brain. As the sixth leading cause of death in the U.S., the disease course usually last from 7 to 10 years on average before the consequential death. In 2019 there are estimated 5.8 million Americans living with AD affecting 16 million family members. At certain stage of the disease course, patients with inability of maintaining their daily functioning highly depend on caregivers, primarily family caregivers, that incur estimated 18.4 billion unpaid hours of cares, which is equivalent to 232 billion dollars. These huge economic burdens and inevitable emotional distress on the family and the society would also increase as the number of AD affected population could triple by 2050. Altered cellular composition is associated with AD progression and decline in cognition, such as neuronal loss and astrocytosis, which is a key feature in neurodegeneration but has often been overlooked in transcriptome research. To explore the cellular composition changes in AD, I developed a deconvolution pipeline for bulk RNA-Seq to account for cell type specific effects in brain tissues. I found that neuronal and astrocyte relative proportions differ between healthy and diseased brains and also among AD cases that carry specific genetic risk variants. Brain carriers of pathogenic mutations in APP, PSEN1, or PSEN2 presented lower neuron and higher astrocyte relative proportions compared to sporadic AD. Similarly, the APOE ε4 allele also showed decreased neuronal and increased astrocyte relative proportions compared to AD non-carriers. In contrast, carriers of variants in TREM2 risk showed a lower degree of neuronal loss compared to matched AD cases in multiple independent studies. These findings suggest that genetic risk factors associated with AD etiology have a specific effect on the cellular composition of AD brains. The digital deconvolution approach provides an enhanced understanding of the fundamental molecular mechanisms underlying neurodegeneration, enabling the analysis of large bulk RNA-sequencing studies for cell composition. It also suggests that correcting for the cellular structure when performing transcriptomic analysis will lead to novel insights of AD. With deconvolution methods to delineate cell population changes in disease condition, it would help interpret transcriptomics results and reveal transcriptional changes in a cell type specific manner. One application demonstrated in this dissertation work is to use cell type proportion as quantitative trait to identify genetic factors associated with cellular composition changes. I performed cell type QTL analysis and identified a common pathway associated with neuronal protection underlying aging brains in the presence or absence of neurodegenerative disease symptoms. A protective variant of TMEM106B, which was previously identified with a protective effect in FTD, was identified to be associated with neuronal proportion in aging brains, suggesting a common pathway underlying neuronal protection and cognitive reservation in elderly. This extended analysis yield from deconvolution results demonstrated one promising direction of using deconvolution followed by cell type QTL analysis in identifying new genes or pathways underlying neurodegenerative or aging brains. To understand the complexity of the brain under disease condition, network analysis as a large-scale system-level approach provides unbiased and data-driven view to identify gene-gene interactions altered by disease status. Using network analysis, I replicated and reconfirmed the co-expression pattern between MS4A gene cluster and TREM2 in sporadic AD, from which further evidence was inferred from Bayesian network analysis to show that MS4A4A might be a potential regulator of TREM2 that is validated by in-vitro experiments. In Autosomal Dominant AD (ADAD) cohort, disrupted and acquired genes were identified from PSEN1 mutation carriers. Among these genes, previously identified AD risk genes and pathways were revealed along with novel findings. These results demonstrated the great potential of applying network approach in identifying disease associated genes and the interactions among them. To conclude the dissertation work from methodological, empirical, and theoretical levels, deconvolution pipeline for bulk RNA-Seq, cell type QTL analysis, and network analysis approaches were applied to understand transcriptome changes underlying disease etiology. From which previous AD related findings were replicated that validated the methods, and novel genes and pathways were identified as potential new therapeutic targets. Based on prior knowledge and empirical evidence observed from this dissertation work, a model is proposed to explain how genetic factors are assembled as a highly interconnected interactome network to affect proteinopathy observed in neurodegenerative disorders, that cause cellular composition changes in the brain, which ultimately leads to cognitive and functional deficits observed in AD patients