Changes in glial cell phenotypes precede overt neurofibrillary tangle formation, correlate with markers of cortical cell damage, and predict cognitive status of individuals at Braak III-IV stages

Clinico-pathological correlation studies show that some otherwise healthy elderly individuals who never developed cognitive impairment harbor a burden of Alzheimer’s disease lesions (plaques and tangles) that would be expected to result in dementia. In the absence of comorbidities explaining such discrepancies, there is a need to identify other brain changes that meaningfully contribute to the cognitive status of an individual in the face of such burdens of plaques and tangles. Glial inflammatory responses, a universal phenomenon in symptomatic AD, show robust association with degree of cognitive impairment, but their significance in early tau pathology stages and contribution to the trajectory of cognitive decline at an individual level remain widely unexplored. We studied 55 brains from individuals at intermediate stages of tau tangle pathology (Braak III-IV) with diverging antemortem cognition (demented vs. non-demented, here termed `resilient’), and age-matched cognitively normal controls (Braak 0-II). We conducted quantitative assessments of amyloid and tau lesions, cellular vulnerability markers, and glial phenotypes in temporal pole (Braak III-IV region) and visual cortex (Braak V-VI region) using artificial-intelligence based semiautomated quantifications. We found distinct glial responses with increased proinflammatory and decreased homeostatic markers, both in regions with tau tangles (temporal pole) and without overt tau deposits (visual cortex) in demented but not in resilient. These changes were significantly associated with markers of cortical cell damage. Similar phenotypic glial changes were detected in the white matter of demented but not resilient and were associated with higher burden of overlying cortical cellular damage in regions with and without tangles. Our data suggest that changes in glial phenotypes in cortical and subcortical regions represent an early phenomenon that precedes overt tau deposition and likely contributes to cell damage and loss of brain function predicting the cognitive status of individuals at intermediate stages of tau aggregate burden (Braak III-IV)

Abeta from APP/PS1 Alzheimer mice hippocampus induced synaptic damage in vivo and in vitro

We aim to investigate the effects of Abeta from young APP/PS1 mouse model of Alzheimer`s disease (AD) on the synaptic integrity, as the loss of synapses strongly correlates with cognitive deficits in patients. Plaque-associated abnormal swellings of neuronal processes represent the first indicator of disease development and might compromise neuronal integrity and synaptic function. Here, we examined the synaptic nature of dystrophic neurites, and the reduction of both synapses and vesicles density in presynaptic terminals along with the progressive accumulation of autophagic structures and Abeta within hippocampal synaptosomes during the aging. We analysed both the direct synaptotoxic effect of plaques in the hippocampus of this model and also the repercussion of the soluble (S1) fraction in neuronal cultures. Hippocampal synapses were observed under both optic and electron microscopy. Synapses and vesicle density were quantified in periplaque and control (plaque-free) areas by electron microscopy. Primary neuronal cultures were incubated for 48 hours with 6-month-old APP/PS1 and wild-type S1 fractions. In addition, Abeta immunodepletion was carried out with different anti-Abeta antibodies and the levels of synaptic proteins were measured by Western-blot (WB). Both synapse number and synaptic-vesicles density were significantly decreased in young APP/PS1 mice, close to the Abeta deposits, in several hippocampal layers. Importantly, there was a correlation between the synaptic deficiencies and the distance to plaques, which presented oligomeric forms in their periphery. Some presynaptic elements were abnormally swollen, containing autophagic vesicles. In addition, we found by WB a decrease in several hippocampal synaptic markers as early as 4 months of age in this model, and also in neuronal cultures incubated with S1 fractions. Significantly, the neuronal reduction in VGLUT was reversed after Abeta immunodepletion. Plaque-associated oligomeric Abeta induced an early deleterious effect on synapses that correlates with memory deficits in young APP/PS1 mice. Moreover, soluble Abeta derived from these transgenic mice reduced synaptic protein content in vitro, which was restored after immunodepletion of Abeta species. Therefore, this model produced synaptotoxic Abeta and may represent a valuable tool to test novel treatments to protect synapses as an early therapeutic approach for AD.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Distinct Microglial Responses in Two Transgenic Murine Models of TAU Pathology

Microglial cells are crucial players in the pathological process of neurodegenerative diseases, such as Alzheimer’s disease (AD). Microglial response in AD has been principally studied in relation to amyloid-beta pathology but, comparatively, little is known about inflammatory processes associated to tau pathology. In the hippocampus of AD patients, where tau pathology is more prominent than amyloid-beta pathology, a microglial degenerative process has been reported. In this work, we have directly compared the microglial response in two different transgenic tau mouse models: ThyTau22 and P301S. Surprisingly, these two models showed important differences in the microglial profile and tau pathology. Where ThyTau22 hippocampus manifested mild microglial activation, P301S mice exhibited a strong microglial response in parallel with high phospho-tau accumulation. This differential phospho-tau expression could account for the different microglial response in these two tau strains. However, soluble (S1) fractions from ThyTau22 hippocampus presented relatively high content of soluble phospho-tau (AT8-positive) and were highly toxic for microglial cells in vitro, whereas the correspondent S1 fractions from P301S mice displayed low soluble phospho-tau levels and were not toxic for microglial cells. Therefore, not only the expression levels but the aggregation of phospho-tau should differ between both models. In fact, most of tau forms in the P301S mice were aggregated and, in consequence, forming insoluble tau species. We conclude that different factors as tau mutations, accumulation, phosphorylation, and/or aggregation could account for the distinct microglial responses observed in these two tau models. For this reason, deciphering the molecular nature of toxic tau species for microglial cells might be a promising therapeutic approach in order to restore the deficient immunological protection observed in AD hippocampus

Tau Oligomer–Containing Synapse Elimination by Microglia and Astrocytes in Alzheimer Disease

Importance: Factors associated with synapse loss beyond amyloid-β plaques and neurofibrillary tangles may more closely correlate with the emergence of cognitive deficits in Alzheimer disease (AD) and be relevant for early therapeutic intervention. // Objective: To investigate whether accumulation of tau oligomers in synapses is associated with excessive synapse elimination by microglia or astrocytes and with cognitive outcomes (dementia vs no dementia [hereinafter termed resilient]) of individuals with equal burdens of AD neuropathologic changes at autopsy. // Design, Setting, and Participants: This cross-sectional postmortem study included 40 human brains from the Massachusetts Alzheimer Disease Research Center Brain Bank with Braak III to IV stages of tau pathology but divergent antemortem cognition (dementia vs resilient) and cognitively normal controls with negligible AD neuropathologic changes. The visual cortex, a region without tau tangle deposition at Braak III to IV stages, was assessed after expansion microscopy to analyze spatial relationships of synapses with microglia and astrocytes. Participants were matched for age, sex, and apolipoprotein E status. Evidence of Lewy bodies, TDP-43 aggregates, or other lesions different from AD neuropathology were exclusion criteria. Tissue was collected from July 1998 to November 2020, and analyses were conducted from February 1, 2022, through May 31, 2023. // Main Outcomes and Measures: Amyloid-β plaques, tau neuropil thread burden, synapse density, tau oligomers in synapses, and internalization of tau oligomer–tagged synapses by microglia and astrocytes were quantitated. Analyses were performed using 1-way analysis of variance for parametric variables and the Kruskal-Wallis test for nonparametric variables; between-group differences were evaluated with Holm-Šídák tests. // Results: Of 40 included participants (mean [SD] age at death, 88 [8] years; 21 [52%] male), 19 had early-stage dementia with Braak stages III to IV, 13 had resilient brains with similar Braak stages III to IV, and 8 had no dementia (Braak stages 0-II). Brains with dementia but not resilient brains had substantial loss of presynaptic (43%), postsynaptic (33%), and colocalized mature synaptic elements (38%) compared with controls and significantly higher percentages of mature synapses internalized by IBA1-positive microglia (mean [SD], 13.3% [3.9%] in dementia vs 2.6% [1.9%] in resilient vs 0.9% [0.5%] in control; P < .001) and by GFAP-positive astrocytes (mean [SD], 17.2% [10.9%] in dementia vs 3.7% [4.0%] in resilient vs 2.7% [1.8%] in control; P = .001). In brains with dementia but not in resilient brains, tau oligomers more often colocalized with synapses, and the proportions of tau oligomer–containing synapses inside microglia (mean [SD] for presynapses, mean [SD], 7.4% [1.8%] in dementia vs 5.1% [1.9%] resilient vs 3.7% [0.8%] control; P = .006; and for postsynapses 11.6% [3.6%] dementia vs 6.8% [1.3%] resilient vs 7.4% [2.5%] control; P = .001) and astrocytes (mean [SD] for presynapses, 7.0% [2.1%] dementia vs 4.3% [2.2%] resilient vs 4.0% [0.7%] control; P = .001; and for postsynapses, 7.9% [2.2%] dementia vs 5.3% [1.8%] resilient vs 3.0% [1.5%] control; P < .001) were significantly increased compared with controls. Those changes in brains with dementia occurred in the absence of tau tangle deposition in visual cortex. // Conclusion and Relevance: The findings from this cross-sectional study suggest that microglia and astrocytes may excessively engulf synapses in brains of individuals with dementia and that the abnormal presence of tau oligomers in synapses may serve as signals for increased glial-mediated synapse elimination and early loss of brain function in AD

Soluble phospho-tau from Alzheimer’s disease hippocampus drives microglial degeneration

The role of microglial cells in the development and progression of Alzheimer’s disease (AD) has not been elucidated. Here, we demonstrated the existence of a weak microglial response in human AD hippocampus which is in contrast to the massive microglial activation observed in APP-based models. Most importantly, microglial cells displayed a prominent degenerative profile (dentate gyrus > CA3 > CA1 > parahippocampal gyrus), including fragmented and dystrophic processes with spheroids, a reduced numerical density, and a significant decrease in the area of surveillance (“microglial domain”). Consequently, there was a substantial decline in the area covered by microglia which may compromise immune protection and, therefore, neuronal survival. In vitro experiments demonstrated that soluble fractions (extracellular/cytosolic) from AD hippocampi were toxic for microglial cells. This toxicity was abolished by AT8 and/or AT100 immunodepletion, validating that soluble phospho-tau was the toxic agent. These results were reproduced using soluble fractions from phospho-tau-positive Thy-tau22 hippocampi. Cultured microglial cells were not viable following phagocytosis of SH-SY5Y cells expressing soluble intracellular phospho-tau. Because the phagocytic capacity of microglial cells is highly induced by apoptotic signals in the affected neurons, we postulate that accumulation of intraneuronal soluble phospho-tau might trigger microglial degeneration in the AD hippocampus. This microglial vulnerability in AD pathology provides new insights into the immunological mechanisms underlying the disease progression and highlights the need to improve or develop new animal models, as the current models do not mimic the microglial pathology observed in the hippocampus of AD patients

Amyloid-β impairs the phagocytosis of dystrophic synapses by astrocytes in Alzheimer's disease.

Reactive astrocytes and dystrophic neurites, most aberrant presynaptic elements, are found surrounding amyloid-β plaques in Alzheimer's disease (AD). We have previously shown that reactive astrocytes enwrap, phagocytose, and degrade dystrophic synapses in the hippocampus of APP mice and AD patients, but affecting less than 7% of dystrophic neurites, suggesting reduced phagocytic capacity of astrocytes in AD. Here, we aimed to gain insight into the underlying mechanisms by analyzing the capacity of primary astrocyte cultures to phagocytose and degrade isolated synapses (synaptoneurosomes, SNs) from APP (containing dystrophic synapses and amyloid-β peptides), Tau (containing AT8- and AT100-positive phosphorylated Tau) and WT (controls) mice. We found highly reduced phagocytic and degradative capacity of SNs-APP, but not AT8/AT100-positive SNs-Tau, as compared with SNs-WT. The reduced astrocyte phagocytic capacity was verified in hippocampus from 12-month-old APP mice, since only 1.60 ± 3.81% of peri-plaque astrocytes presented phagocytic structures. This low phagocytic capacity did not depend on microglia-mediated astrocyte reactivity, because removal of microglia from the primary astrocyte cultures abrogated the expression of microglia-dependent genes in astrocytes, but did not affect the phagocytic impairment induced by oligomeric amyloid-β alone. Taken together, our data suggest that amyloid-β, but not hyperphosphorylated Tau, directly impairs the capacity of astrocytes to clear the pathological accumulation of oligomeric amyloid-β, as well as of peri-plaque dystrophic synapses containing amyloid-β, perhaps by reducing the expression of phagocytosis receptors such as Mertk and Megf10, thus increasing neuronal damage in AD. Therefore, the potentiation or recovery of astrocytic phagocytosis may be a novel therapeutic avenue in AD

Plaque-Associated Oligomeric Amyloid-Beta Drives Early Synaptotoxicity in APP/PS1 Mice Hippocampus: Ultrastructural Pathology Analysis

Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by initial memory impairments that progress to dementia. In this sense, synaptic dysfunction and loss have been established as the pathological features that best correlate with the typical early cognitive decline in this disease. At the histopathological level, post mortem AD brains typically exhibit intraneuronal neurofibrillary tangles (NFTs) along with the accumulation of amyloid-beta (Abeta) peptides in the form of extracellular deposits. Specifically, the oligomeric soluble forms of Abeta are considered the most synaptotoxic species. In addition, neuritic plaques are Abeta deposits surrounded by activated microglia and astroglia cells together with abnormal swellings of neuronal processes named dystrophic neurites. These periplaque aberrant neurites are mostly presynaptic elements and represent the first pathological indicator of synaptic dysfunction. In terms of losing synaptic proteins, the hippocampus is one of the brain regions most affected in AD patients. In this work, we report an early decline in spatial memory, along with hippocampal synaptic changes, in an amyloidogenic APP/PS1 transgenic model. Quantitative electron microscopy revealed a spatial synaptotoxic pattern around neuritic plaques with significant loss of periplaque synaptic terminals, showing rising synapse loss close to the border, especially in larger plaques. Moreover, dystrophic presynapses were filled with autophagic vesicles in detriment of the presynaptic vesicular density, probably interfering with synaptic function at very early synaptopathological disease stages. Electron immunogold labeling showed that the periphery of amyloid plaques, and the associated dystrophic neurites, was enriched in Abeta oligomers supporting an extracellular location of the synaptotoxins. Finally, the incubation of primary neurons with soluble fractions derived from 6-month-old APP/PS1 hippocampus induced significant loss of synaptic proteins, but not neuronal death. Indeed, this preclinical transgenic model could serve to investigate therapies targeted at initial stages of synaptic dysfunction relevant to the prodromal and early AD.This study was supported by the Instituto de Salud Carlos III (ISCiii) of Spain, co-financed by the FEDER funds from European Union, through grants PI18/01557 (to AG) and PI18/01556 (to JV); by the Junta de Andalucia Consejería de Economía y Conocimiento through grants UMA18-FEDERJA-211 (to AG), P18-RT-2233 (to AG), and US-1262734 (to JV) co-financed by Programa Operativo FEDER 2014–2020; by the Spanish Minister of Science and Innovation grant PID2019-108911RA-100 (to DB-V), Beatriz Galindo program BAGAL18/00052 (to DB-V) grant PID2019-107090RA-I00 (to IM-G), and Ramon y Cajal Program RYC-2017-21879 (to IM-G); and by the Malaga University grants B1-2019_07 (to ES-M) and B1-2019_06 (to IM-G). MM-O held a predoctoral contract from Malaga University and ES-M a postdoctoral contract (DOC_00251) from Junta de Andalucia.Ye

Additional file 1 of Real-time imaging of mitochondrial redox reveals increased mitochondrial oxidative stress associated with amyloid β aggregates in vivo in a mouse model of Alzheimer’s disease

Additional file 1: Fig. S1. Validation of AAV.hSyn.mt-roGFP in vitro. a. Mitochondrial co-transfection verified proper targeting of mt-roGFP to mitochondria. N2a cells (top) and primary cortical neurons (bottom) were co-transfected with mt-roGFP (green) and mRuby-Mito-7 (red) and subjected to confocal microscopy imaging. Scale bar represents 10 μm. b. Double immunolabelling of mt-roGFP (green) and mRuby-ER5 (red, targeting endoplasmic reticulum, ER) in N2a cells shows lack of colocalization and supports the mitochondrial localization of mt-roGFP. Scale bar represents 10 μm. c. In vitro imaging of cellular oxidative stress with mt-roGFP. Primary cortical neurons were exposed to either the oxidant DTDP or the reducing agent DTT. Images at 800 nm (red), 900 nm (green) and merged are shown. d. The relative changes in ratio 800/900 were represented by histograms of ratio 800/900 frequency distribution in control conditions (grey) and 20 min after exposure to DTT 1 mM (blue) and DTDP 100 μM (red) (Control, n = 143 cells; DTT 1 mM, n = 125; DTDP 100 μM, n = 109 cells). Fig. S2. Validation of pAAV.hSyn.mt-roGFP ex vivo. AAV.hSyn.mt-roGFP targets neuronal mitochondria in vivo. a. Colocalization of AAV.hSyn.mt-roGFP (green), NeuN (red) and GS (glutamine synthetase, magenta) in the mouse cortex shown by immunohistochemistry. Note that AAV.hSyn.mt-roGFP only colocalizes with the neuronal marker NeuN. Scale bar represents 10 μm. b. Colocalization of AAV.hSyn.mt-roGFP (green), HSP60 (mitochondrial marker, red) and NeuN (magenta) in the cortex shown by immunohistochemistry. Scale bar represents 10 μm. c. Inset. Colocalization of AAV.hSyn.mt-roGFP (green) and HSP60 (red) in cortex shown by immunohistochemistry (top). Scale bar 5 μm. Graph shows intensity profile of the ROI across the cell. Green line represents the fluorescence intensity of AAV.hSyn.mt-roGFP and red line represents the fluorescence intensity of HSP60. Fig. S3. Original images excited at 800nm and 900nm of Fig. 1b. Fig. S4. Original images excited at 800nm and 900nm of Fig. 2b. Fig. S5. Mitochondrial oxidative stress in male and female mice. Mitochondrial oxidative stress (Ratio 800/900) in neurons was compared between non-Tg and APP/PS1 Tg mice at 10 months of age within males (a) or females (b). Note that only for males the difference is significantly different (a. Males: average per field of view: non-Tg: 0.95 ± 0.026, n = 31 z-stacks; APP/PS1: 1.17 ± 0.046, n = 41 z-stacks from 5 and 9 mice respectively, ***p = 0.0001; Average per mouse: non-Tg: 0.95 ± 0.037, n = 5 mice; APP/PS1: 1.19 ± 0.073, n = 9 mice, *p=0.0190. b. Females: average per field of view: non-Tg: 1.038 ± 0.038, n = 38 z-stacks; APP/PS1: 1.17 ± 0.043, n = 19 z-stacks from 6 and 3 mice respectively; Average per mouse: non-Tg: 1.02 ± 0.08, n = 6 mice; APP/PS1: 1.19 ± 0.067, n = 3 mice). Error bars represent mean ± SEM. Fig. S6. The overall mitochondrial redox levels are not elevated in AD transgenic mouse neurons before Aβ plaque deposition. a. In vivo images of neurites and cell bodies expressing pAAV.hSyn.mt-roGFP in mitochondria in non-Tg (top) and APP/PS1 Tg mice (bottom) in young mice. Scale bar represents 10 μm. b, c. Scatter dot plot represents overall mitochondrial oxidative stress (Ratio 800/900) in non-Tg and APP/PS1 Tg mice at 3 months of age, before plaque deposition, in mitochondria in neurons (b, average per field of view, non-Tg: 0.83 ± 0.024, n = 18 z-stacks from 3 mice (3 male); APP/PS1: 0.87 ± 0.024, n = 42 z-stacks from 6 mice (3 male, 3 female); c. average per mouse, non-Tg: 0.82 ± 0.039, n = 3 mice (3 male); APP/PS1: 0.87 ± 0.034, n = 6 mice (3 male, 3 female)). Error bars represent mean ± SEM. Blue dots denote male and pink dots denote female. d. Histogram of mitochondrial oxidative stress frequency distribution (indicated by Ratio 800/900) in the young non-Tg and APP/PS1 Tg mice. e. Representative high resolution pseudocolor images of somas (top) and neurites (bottom) expressing AAV.hSyn.mt-roGFP in mitochondria in vivo in young non-Tg (left) and APP/PS1 Tg mice (right). Scale bar represents 15 or 10 μm. f. Comparison of mitochondrial oxidative stress (Ratio 800/900) within somas or neurites in 3-month-old non-Tg and APP/PS1 Tg mice. APP/PS1 Tg mice showed higher oxidative stress levels in mitochondria in neurites. Error bars represent mean ± SEM. (somas: 0.79 ± 0.023, n = 9 z-stacks from 3 non-Tg mice (3 male), and 0.75 ± 0.026, n = 10 z-stacks from 3 APP/PS1 Tg mice (1 male, 2 females); neurites: 0.82 ± 0.040, n = 9 z-stacks from 3 non-Tg mice (3 male), and 0.92 ± 0.030, n = 10 z-stacks from 3 APP/PS1 Tg mice (1 males, 4 females); *p = 0.0467). g. Comparison of mitochondrial oxidative stress (Ratio 800/900) in the different cell compartments (somas and neurites) in 3-month-old (old) non-Tg and APP/PS1 Tg mice. Neurites showed significantly higher oxidative stress levels in mitochondria in the APP/PS1 Tg mouse when compared to the somas. Error bars represent mean ± SEM. (Young non-Tg: 0.79 ± 0.023 for somas and 0.82 ± 0.040 for neurites, n = 9 z-stacks from 3 mice (3 male); Young APP/PS1: 0.75 ± 0.026 for somas and 0.92 ± 0.030 for neurites, n = 10 z-stacks from 3 mice (1 male, 2 female), ***p = 0.0003). Blue dots denote male and pink dots denote female. Fig. S7. Original images excited at 800nm and 900nm of Fig. 3b. Fig. S8. Original images excited at 800nm and 900nm of Fig. 4a. Fig. S9. Original images excited at 800nm and 900nm of Fig. 5a. Fig. S10. SS31 reduces Aβ-associated dystrophic neurite number but not amyloid burden in the AD transgenic mouse. a.  Representative images of the global amount of amyloid in the cortex of SS31 and SS20 treated APP/PS1 mice at 10 mo of age after Aβ immunostaining. Scale bar represents 100 μm.  b. Scatter dot plots represent the quantification of amyloid load in the cortex after anti-Aβ immunostaining or ThioS labeling. The number of dense-core plaques detected by ThioS (top) and the overall load of Aβ (bottom) was comparable among SS31 and SS20 APP/PS1 treated mice. n = 7 mice per condition. Histograms represent the dense core plaque (top) and diffuse amyloid deposit (bottom) size in both conditions. c. Representative images of neuritic dystrophies (arrow heads, neurofilaments in green) around amyloid plaques (blue) in APP/PS1 mouse after either SS31 or SS20 treatment. Scale bar 20 μm. d. Scatter dot plot represents the quantification of the number of dystrophic neurites observed per plaque, n = 362 plaques from 4 SS31 APP/PS1 treated mice and n = 295 plaques from 4 SS31 APP/PS1 treated mice, **p < 0.05. e. Scatter dot plot represents the percentage of plaques showing dystrophic neurites, n = 4 – 5 areas per 4 mouse per condition, *p = 0.022

Developmental outcome of electroencephalographic findings in SYNGAP1 encephalopathy

SYNGAP1 haploinsufficiency results in a developmental and epileptic encephalopathy (DEE) causing generalized epilepsies accompanied by a spectrum of neurodevelopmental symptoms. Concerning interictal epileptiform discharges (IEDs) in electroencephalograms (EEG), potential biomarkers have been postulated, including changes in background activity, fixation-off sensitivity (FOS) or eye closure sensitivity (ECS). In this study we clinically evaluate a new cohort of 36 SYNGAP1-DEE individuals. Standardized questionnaires were employed to collect clinical, electroencephalographic and genetic data. We investigated electroencephalographic findings, focusing on the cortical distribution of interictal abnormalities and their changes with age. Among the 36 SYNGAP1-DEE cases 18 presented variants in the SYNGAP1 gene that had never been previously reported. The mean age of diagnosis was 8 years and 8 months, ranging from 2 to 17 years, with 55.9% being male. All subjects had global neurodevelopmental/language delay and behavioral abnormalities; 83.3% had moderate to profound intellectual disability (ID), 91.7% displayed autistic traits, 73% experienced sleep disorders and 86.1% suffered from epileptic seizures, mainly eyelid myoclonia with absences (55.3%). A total of 63 VEEGs were revised, observing a worsening of certain EEG findings with increasing age. A disorganized background was observed in all age ranges, yet this was more common among older cases. The main IEDs were bilateral synchronous and asynchronous posterior discharges, accounting for ≥50% in all age ranges. Generalized alterations with maximum amplitude in the anterior region showed as the second most frequent IED (≥15% in all age ranges) and were also more common with increasing age. Finally, diffuse fast activity was much more prevalent in cases with 6 years or older. To the best of our knowledge, this is the first study to analyze EEG features across different age groups, revealing an increase in interictal abnormalities over infancy and adolescence. Our findings suggest that SYNGAP1 haploinsufficiency has complex effects in human brain development, some of which might unravel at different developmental stages. Furthermore, they highlight the potential of baseline EEG to identify candidate biomarkers and the importance of natural history studies to develop specialized therapies and clinical trials