Dynamic microglia alterations associate with hippocampal network impairments: A turning point in amyloid pathology progression.

Alzheimer's disease is a progressive neurological disorder causing memory loss and cognitive decline. The underlying causes of cognitive deterioration and neurodegeneration remain unclear, leading to a lack of effective strategies to prevent dementia. Recent evidence highlights the role of neuroinflammation, particularly involving microglia, in Alzheimer's disease onset and progression. Characterizing the initial phase of Alzheimer's disease can lead to the discovery of new biomarkers and therapeutic targets, facilitating timely interventions for effective treatments. We used the AppNL-G-F knock-in mouse model, which resembles the amyloid pathology and neuroinflammatory characteristics of Alzheimer's disease, to investigate the transition from a pre-plaque to an early plaque stage with a combined functional and molecular approach. Our experiments show a progressive decrease in the power of cognition-relevant hippocampal gamma oscillations during the early stage of amyloid pathology, together with a modification of fast-spiking interneuron intrinsic properties and postsynaptic input. Consistently, transcriptomic analyses revealed that these effects are accompanied by changes in synaptic function-associated pathways. Concurrently, homeostasis- and inflammatory-related microglia signature genes were downregulated. Moreover, we found a decrease in Iba1-positive microglia in the hippocampus that correlates with plaque aggregation and neuronal dysfunction. Collectively, these findings support the hypothesis that microglia play a protective role during the early stages of amyloid pathology by preventing plaque aggregation, supporting neuronal homeostasis, and overall preserving the oscillatory network's functionality. These results suggest that the early alteration of microglia dynamics could be a pivotal event in the progression of Alzheimer's disease, potentially triggering plaque deposition, impairment of fast-spiking interneurons, and the breakdown of the oscillatory circuitry in the hippocampus.

A cell autonomous regulator of neuronal excitability modulates tau in Alzheimer's disease vulnerable neurons.

Neurons from layer II of the entorhinal cortex (ECII) are the first to accumulate tau protein aggregates and degenerate during prodromal Alzheimer's disease (AD). Gaining insight into the molecular mechanisms underlying this vulnerability will help reveal genes and pathways at play during incipient stages of the disease. Here, we use a data-driven functional genomics approach to model ECII neurons in silico and identify the proto-oncogene DEK as a regulator of tau pathology. We show that epigenetic changes caused by Dek silencing alter activity-induced transcription, with major effects on neuronal excitability. This is accompanied by gradual accumulation of tau in the somatodendritic compartment of mouse ECII neurons in vivo, reactivity of surrounding microglia, and microglia-mediated neuron loss. These features are all characteristic of early AD. The existence of a cell-autonomous mechanism linking AD pathogenic mechanisms in the precise neuron type where the disease starts provides unique evidence that synaptic homeostasis dysregulation is of central importance in the onset of tau pathology in AD.

Molecular Mechanisms of Amyloid-ß Self-Assembly Seeded by In Vivo-Derived Fibrils and Inhibitory Effects of the BRICHOS Chaperone.

Self-replication of amyloid-ß-peptide (Aß) fibril formation is a hallmark in Alzheimer's disease (AD). Detailed insights have been obtained in Aß self-assembly in vitro, yet whether similar mechanisms are relevant in vivo has remained elusive. Here, we investigated the ability of in vivo-derived Aß fibrils from two different amyloid precursor protein knock-in AD mouse models to seed Aß42 aggregation, where we quantified the microscopic rate constants. We found that the nucleation mechanism of in vivo-derived fibril-seeded Aß42 aggregation can be described with the same kinetic model as that in vitro. Further, we identified the inhibitory mechanism of the anti-amyloid BRICHOS chaperone on seeded Aß42 fibrillization, revealing a suppression of secondary nucleation and fibril elongation, which is strikingly similar as observed in vitro. These findings hence provide a molecular understanding of the Aß42 nucleation process triggered by in vivo-derived Aß42 propagons, providing a framework for the search for new AD therapeutics.

Targeting galectin-3 to counteract spike-phase uncoupling of fast-spiking interneurons to gamma oscillations in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a progressive multifaceted neurodegenerative disorder for which no disease-modifying treatment exists. Neuroinflammation is central to the pathology progression, with evidence suggesting that microglia-released galectin-3 (gal3) plays a pivotal role by amplifying neuroinflammation in AD. However, the possible involvement of gal3 in the disruption of neuronal network oscillations typical of AD remains unknown. METHODS: Here, we investigated the functional implications of gal3 signaling on experimentally induced gamma oscillations ex vivo (20-80 Hz) by performing electrophysiological recordings in the hippocampal CA3 area of wild-type (WT) mice and of the 5×FAD mouse model of AD. In addition, the recorded slices from WT mice under acute gal3 application were analyzed with RT-qPCR to detect expression of some neuroinflammation-related genes, and amyloid-ß (Aß) plaque load was quantified by immunostaining in the CA3 area of 6-month-old 5×FAD mice with or without Gal3 knockout (KO). RESULTS: Gal3 application decreased gamma oscillation power and rhythmicity in an activity-dependent manner, which was accompanied by impairment of cellular dynamics in fast-spiking interneurons (FSNs) and pyramidal cells. We found that the gal3-induced disruption was mediated by the gal3 carbohydrate-recognition domain and prevented by the gal3 inhibitor TD139, which also prevented Aß42-induced degradation of gamma oscillations. Furthermore, the 5×FAD mice lacking gal3 (5×FAD-Gal3KO) exhibited WT-like gamma network dynamics and decreased Aß plaque load. CONCLUSIONS: We report for the first time that gal3 impairs neuronal network dynamics by spike-phase uncoupling of FSNs, inducing a network performance collapse. Moreover, our findings suggest gal3 inhibition as a potential therapeutic strategy to counteract the neuronal network instability typical of AD and other neurological disorders encompassing neuroinflammation and cognitive decline.

S100A9 amyloid growth and S100A9 fibril-induced impairment of gamma oscillations in area CA3 of mouse hippocampus ex vivo is prevented by Bri2 BRICHOS.

The pro-inflammatory and highly amyloidogenic protein S100A9 is central to the amyloid-neuroinflammatory cascade in neurodegenerative diseases leading to cognitive impairment. Molecular chaperone activity of Bri2 BRICHOS has been demonstrated against a range of amyloidogenic polypeptides. Using a combination of thioflavin T fluorescence kinetic assay, atomic force microscopy and immuno electron microscopy we show here that recombinant Bri2 BRICHOS effectively inhibits S100A9 amyloid growth by capping amyloid fibrils. Using ex-vivo neuronal network electrophysiology in mouse brain slices we also show that both native S100A9 and amyloids of S100A9 disrupt cognition-relevant gamma oscillation power and rhythmicity in hippocampal area CA3 in a time- and protein conformation-dependent manner. Both effects were associated with Toll-like receptor 4 (TLR4) activation and were not observed upon TLR4 blockade. Importantly, S100A9 that had co-aggregated with Bri2 BRICHOS did not elicit degradation of gamma oscillations. Taken together, this work provides insights on the potential influence of S100A9 on cognitive dysfunction in Alzheimer's disease (AD) via gamma oscillation impairment from experimentally-induced gamma oscillations, and further highlights Bri2 BRICHOS as a chaperone against detrimental effects of amyloid self-assembly.

Functionally-distinct pyramidal cell subpopulations during gamma oscillations in mouse hippocampal area CA3.

Gamma oscillations (γ-oscillations) in hippocampal area CA3 are essential for memory function. Particularly, CA3 is involved in the memory related process pattern completion, which is linked with the γ-oscillations in human hippocampus. Recent studies suggest that heterogeneity in the functional properties of pyramidal cells (PCs) in CA3 plays an important role in hippocampal function. By performing concomitant recordings of PC activity and network γ-oscillations in CA3 we found three functionally-different PC subpopulations. PCs with high spike-frequency adaptation (hAPC) have the strongest action potential gamma phase-coupling, PCs with low adaptation (lAPC) show lower phase-coupling and PCs displaying a burst-firing pattern (BPC) remained quiescent. In addition, we discovered that hAPC display the highest excitatory/inhibitory drive, followed by lAPC, and lastly BPC. In conclusion, our data advance the hypothesis that PCs in CA3 are organized into subpopulations with distinct functional roles for cognition-relevant network dynamics and provide new insights in the physiology of hippocampus.

Impaired spike-gamma coupling of area CA3 fast-spiking interneurons as the earliest functional impairment in the AppNL-G-F mouse model of Alzheimer's disease.

In Alzheimer's disease (AD) the accumulation of amyloid-ß (Aß) correlates with degradation of cognition-relevant gamma oscillations. The gamma rhythm relies on proper neuronal spike-gamma coupling, specifically of fast-spiking interneurons (FSN). Here we tested the hypothesis that decrease in gamma power and FSN synchrony precede amyloid plaque deposition and cognitive impairment in AppNL-G-F knock-in mice (AppNL-G-F). The aim of the study was to evaluate the amyloidogenic pathology progression in the novel AppNL-G-F mouse model using in vitro electrophysiological network analysis. Using patch clamp of FSNs and pyramidal cells (PCs) with simultaneous gamma oscillation recordings, we compared the activity of the hippocampal network of wild-type mice (WT) and the AppNL-G-F mice at four disease stages (1, 2, 4, and 6 months of age). We found a severe degradation of gamma oscillation power that is independent of, and precedes Aß plaque formation, and the cognitive impairment reported previously in this animal model. The degradation correlates with increased Aß1-42 concentration in the brain. Analysis on the cellular level showed an impaired spike-gamma coupling of FSN from 2 months of age that correlates with the degradation of gamma oscillations. From 6 months of age PC firing becomes desynchronized also, correlating with reports in the literature of robust Aß plaque pathology and cognitive impairment in the AppNL-G-F mice. This study provides evidence that impaired FSN spike-gamma coupling is one of the earliest functional impairment caused by the amyloidogenic pathology progression likely is the main cause for the degradation of gamma oscillations and consequent cognitive impairment. Our data suggests that therapeutic approaches should be aimed at restoring normal FSN spike-gamma coupling and not just removal of Aß.

Amphetamine sensitization alters hippocampal neuronal morphology and memory and learning behaviors.

It is known that continuous abuse of amphetamine (AMPH) results in alterations in neuronal structure and cognitive behaviors related to the reward system. However, the impact of AMPH abuse on the hippocampus remains unknown. The aim of this study was to determine the damage caused by AMPH in the hippocampus in an addiction model. We reproduced the AMPH sensitization model proposed by Robinson et al. in 1997 and performed the novel object recognition test (NORt) to evaluate learning and memory behaviors. After the NORt, we performed Golgi-Cox staining, a stereological cell count, immunohistochemistry to determine the presence of GFAP, CASP3, and MT-III, and evaluated oxidative stress in the hippocampus. We found that AMPH treatment generates impairment in short- and long-term memories and a decrease in neuronal density in the CA1 region of the hippocampus. The morphological test showed an increase in the total dendritic length, but a decrease in the number of mature spines in the CA1 region. GFAP labeling increased in the CA1 region and MT-III increased in the CA1 and CA3 regions. Finally, we found a decrease in Zn concentration in the hippocampus after AMPH treatment. An increase in the dopaminergic tone caused by AMPH sensitization generates oxidative stress, neuronal death, and morphological changes in the hippocampus that affect cognitive behaviors like short- and long-term memories.

Modulation of Kv3.1/Kv3.2 promotes gamma oscillations by rescuing Aß-induced desynchronization of fast-spiking interneuron firing in an AD mouse model in vitro.

KEY POINTS: Gamma oscillations (30-80 Hz) are important for cognitive functions and depend on the synchronized activity of fast-spiking interneurons (FSN), which is crucial for network stability. Gamma oscillations are degraded in Alzheimer's disease (AD) patients exhibiting cognitive impairment, with the degree of cognitive decline correlating with the severity of gamma disruption in response to neurotoxic amyloid-beta peptide (Aß). Small molecule compounds EX15 and RE01 modulate Kv3.1/Kv3.2 potassium channels on FSN, resulting in faster activation kinetics and increased firing frequency, suggesting direct consequences for cognition-relevant gamma oscillations, particularly in a situation where network activity is pathologically compromised in the presence of neurotoxic Aß. Using electrophysiological techniques in an in vitro AD model, we found a significant effect of EX15 and RE01 with respect to counteracting toxic Aß effects on neuronal dynamics, advocating for targeting FSN activity to rescue cognitive performance from impairment caused by neurodegenerative triggers. ABSTRACT: Rhythmic electrical activity in neuronal networks such as gamma oscillations (30-80 Hz) underlies cognitive functions such as sensory perception, attention and memory. Gamma oscillations are disrupted in Alzheimer's disease (AD) patients and animal AD models, with the severity of cognitive decline correlating with the degree of rhythm disruption. Misfolded amyloid-ß peptide (Aß) is assumed to be a key trigger of AD pathology and has been show to de-synchronize action potential firing in fast-spiking interneurons (FSN), which is crucial for entraining neuronal network activity into the gamma rhythm. The synchronizing activity of FSN therefore has become one of the most suitable targets to counteract disease-driven degradation of gamma oscillations and consequent cognitive decline. EX15 and RE01 are small-molecule compounds that modulate Kv3.1/Kv3.2 potassium channels, resulting in faster activation kinetics and increased FSN firing frequency. In the present study, we investigated the potential pro-cognitive effects of EX15 and RE01 by testing their ability to modulate FSN activity during ongoing gamma oscillations in normal and Aß-disrupted network states in mouse hippocampus in vitro. In the compromised, but not the uncompromised, network state with gamma oscillations partially disrupted by Aß, both compounds improve gamma oscillation regularity by promoting re-synchronization of FSN action potential firing. Our data suggest a therapeutic potential for compounds such as EX15 and RE01, which can rescue normal action potential firing parameters in FSN, in the search for disease-modifying drug candidates counteracting the progressive dysfunction of neuronal network dynamics that underlies the cognitive impairment typical of AD and other cognitive brain disorders.

Apomorphine effects on the hippocampus.

Apomorphine is a non-specific dopamine receptor agonist that has been used in the treatment of some diseases and mental disorders. Its use has particularly well documented in Parkinson's disease (PD). The dopaminergic agonists like apomorphine are related to oxidative processes that could induce cell damage and the functional impairment of some structures in the brain. However, most information about apomorphine in literature is focused on the improvement of the motor problems characteristic of PD, but little is known about the effects on cognitive behaviors and brain structures indirectly related to motor function. The presence of dopaminergic receptors in the hippocampus has recently been discovered, in connection with cognitive behaviors like learning and memory, these receptors are needed in neuronal plasticity. There has been a growing interest to know if this structure could be compromised by the effect of apomorphine and elucidate if part of the cognitive impairment present in the PD is due to the effect of apomorphine. In this mini-review, we summarized how apomorphine has been used since its creation, we discuss the latest information about its effect on the hippocampus and also the future perspectives to fully understand the effects of this compound.

The Effects of Non-selective Dopamine Receptor Activation by Apomorphine in the Mouse Hippocampus.

Apomorphine is a dopamine receptor agonist that activates D1-D5 dopamine receptors and that is used to treat Parkinson's disease (PD). However, the effect of apomorphine on non-motor activity has been poorly studied, and likewise, the effects of dopaminergic activation in brain areas that do not fulfill motor functions are unclear. The aim of this study was to determine how dopamine receptor activation affects behavior, as well as plasticity, morphology, and oxidative stress in the hippocampus. Adult mice were chronically administered apomorphine (1 mg/kg for 15 days), and the effects on memory and learning, synaptic plasticity, dendritic length, inflammatory responses, and oxidative stress were evaluated. Apomorphine impaired learning and long-term memory in mice, as evaluated in the Morris water maze test. In addition, electrophysiological recording of field excitatory postsynaptic potentials (fEPSP) indicated that the long-term potentiation (LTP) of synaptic transmission in the CA1 region of the hippocampus was fully impaired by apomorphine. In addition, a Sholl analysis of Golgi-Cox stained neurons showed that apomorphine reduced the total length of dendrites in the CA1 region of the hippocampus. Finally, there were more reactive astrocytes and oxidative stress biomarkers in mice administered apomorphine, as measured by GFAP immunohistochemistry and markers of redox balance, respectively. Hence, the non-selective activation of dopaminergic receptors in the hippocampus by apomorphine triggers deficiencies in learning and memory, it prevents LTP, reduces dendritic length, and provokes neuronal damage.

The effects of amphetamine exposure on juvenile rats on the neuronal morphology of the limbic system at prepubertal, pubertal and postpubertal ages.

Amphetamines (AMPH) are psychostimulants widely used for therapy as well as for recreational purposes. Previous results of our group showed that AMPH exposure in pregnant rats induces physiological and behavioral changes in the offspring at prepubertal and postpubertal ages. In addition, several reports have shown that AMPH are capable of modifying the morphology of neurons in some regions of the limbic system. These modifications can cause some psychiatric conditions. However, it is still unclear if there are changes to behavioral and morphological levels when low doses of AMPH are administered at a juvenile age. The aim of this study was to assess the effect of AMPH administration (1mg/kg) in Sprague-Dawley rats (postnatal day, PD21-PD35) on locomotor activity in a novel environment and compare the neuronal morphology of limbic system areas at three different ages: prepubertal (PD 36), pubertal (PD50) and postpubertal (PD 62). We found that AMPH altered locomotor activity in the prepubertal group, but did not have an effect on the other two age groups. The Golgi-Cox staining method was used to describe the neural morphology of five limbic regions: (Layers 3 and 5) the medial prefrontal cortex (mPFC), the dorsal and ventral hippocampus, the nucleus accumbens and the amygdala, showing that AMPH induced changes at pubertal ages in arborization and spine density of these neurons, but interestingly these changes did not persist at postpubertal ages. Our findings suggest that even early-life AMPH exposure does not induce long-term behavioral and morphological changes, however it causes alterations at pubertal ages in the limbic system networks, a stage of life strongly associated with the development of substance abuse behaviors.

Combined administration of cerebrolysin and donepezil induces plastic changes in prefrontal cortex in aged mice.

Cerebrolysin (Cbl) shows neurotrophic and neuroprotective properties while donepezil (Dnp) is a potent acetylcholinesterase (AChE) inhibitor, both drugs are prescribed for Alzheimer's disease (AD) treatment. Previous studies have shown that the Dnp and Cbl administered separately, modify dendritic morphology of neurons in the prefrontal cortex and hippocampus in senile rodents. Since the deficit of neurotrophic factor activity is implicated in the degeneration of cholinergic neurons of basal forebrain, a combination therapy of Dnp and Cbl has been tested recently in Alzheimer's patients. However, the plastic changes that may underlie this combined treatment have not yet been explored. We present here the effect of the combined administration of Cbl and Dnp on dendritic morphology in brain regions related to learning and memory in aged mice. The Golgi-Cox staining protocol and Sholl analysis were used for studying dendritic changes. Cbl and Dnp were administrated daily for 2 months to 9-months-old mice. Locomotor activity was assessed, as well as the dendritic morphology of neurons in several limbic regions was analyzed. Results showed that Cbl and Dnp induced an increase in locomotor activity without synergistic effect. The Cbl or Dnp treatment modified the dendritic morphology of neurons from prefrontal cortex (PFC), dorsal hippocampus (DH), dentate gyrus (DG), and the shell of nucleus accumbens (NAcc). These changes show an increase in the total dendritic length and spine density, resulting in an improvement of dendritic arborization. Prominently, a synergistic effect of Cbl and Dnp was observed on branching order and total dendritic length of pyramidal neurons from PFC. These results suggest that Dnp and Cbl may induce plastic changes in a manner independent of each other, but could enhance their effect in target cells from PFC.