Abstract Body

Alzheimer's disease is associated with a loss of synapses that correlates with the decline in cognition observed in patients. In my presentation, I will discuss our recent studies on the role of the genes associated with familial Alzheimer's disease, APP and presenilins, and with sporadic Alzheimer's disease, ApoE, in synaptic function. My presentation will be purely directed towards a molecular and cellular understanding of the functions of these genes with no claims of relevance for Alzheimer's disease apart from the fact that these genes are strongly associated with Alzheimer's disease, suggesting that a basic understanding of their roles in brain may help to gain insight into how Alzheimer's disease develops.

Aims

Synaptic loss and cytoskeletal abnormalities, such as cofilin-actin rods, are biological hallmarks of Alzheimer's disease (AD).

We have recently demonstrated that the actin-binding protein CAP2 is a master regulator of cofilin in the synapse. The formation of Cys32-dependent CAP2 dimers can control cofilin synaptic availability in plasticity processes that are crucial for memory. CAP2 is down-regulated in AD and the reduced CAP2 availability impairs CAP2/cofilin association.

Considering the relevance of CAP2 in controlling cofilin-function, the aim of the study was to investigate CAP2 role in synaptic loss and in the generation of rods in AD.

Methods

We used APP/PS1 mice as animal model of AD. We exposed primary hippocampal cultures to Aβ oligomers for 24 hours to model synaptic loss and actin rods formation in vitro.

Results

We found that CAP2 overexpression in AD mice prevents cognitive decline and actin rods formation. Furthermore, restoring CAP2 levels rescues the altered synaptic availability of cofilin because it increases CAP2 dimer levels.

In neuronal cultures, taking advantage of 3D confocal analysis, we found that CAP2 accumulates in actin rods, when neurons were specifically exposed to Aβ oligomers. In the synapses, cofilin is reduced and CAP2 dimerization impaired. In hippocampal neuronal cultures, CAP2 overexpression prevents cofilin-actin rods formation and synapses loss induced by Aβ oligomers. We demonstrated that Cys32 dependent CAP2 dimers are important for CAP2 role in the spines but are not essential for actin rods formation.

Conclusions

Overall, our data support the involvement of cofilin/CAP2 pathway in the generation of cytoskeleton abnormalities and in synaptic dysfunction in AD. As an early event in the neurodegenerative cascade, cofilin/CAP2 pathway is an ideal target for therapeutic intervention that might be useful in treatment of AD.

Aims

Synapses can modify their strength in response to activity, and the unique properties of synapses that regulate their plasticity are essential for memory. Long-term potentiation (LTP) is considered the physiological basis for how neurons encode new memories. LTP is associated with memory deficits in tauopathy models, but the mechanism by which pathogenic tau inhibits synaptic plasticity is unknown. Here, our objective was to delineate the mechanism by which pathogenic tau inhibits LTP in neurons underlying tauopathy-related memory loss.

Methods

To study synaptic plasticity in human induced pluripotent stem cell (iPSC)-derived neurons, we used a protocol to induce NMDA receptor-dependent LTP measured by electrophysiology and by immunolabeling to monitor the trafficking of postsynaptic AMPA receptors (AMPARs). We also used techniques to monitor the local translation in dendrites after LTP induction. To determine the impact of pathogenic tau on LTP, human iPSC-derived neurons carrying tau mutations that cause frontotemporal dementia were compared to isogenic control human iPSC-derived neurons. The transgenic PS19 tauopathy mouse model was used for electrophysiological and behavioral studies to investigate the mechanism underlying the tau-mediated plasticity impairment.

Results

We found that pathogenic tau inhibited postsynaptic AMPARs delivery for LTP expression in human neurons. In control neurons, local protein synthesis occurred after LTP induction, and protein synthesis was required for LTP expression. Pathogenic tau obstructed plasticity by blocking activity-dependent protein synthesis in dendrites after LTP induction. Interestingly, tau binds to a translation initiation factor that regulates activity-dependent protein synthesis in neurons.

Conclusions

This work revealed that pathogenic tau directly disrupts the expression of LTP at synapses by blocking activity-dependent translation in dendrites. The mechanism by which tau inhibits protein synthesis during LTP involves a direct interaction of the tau protein with a translation initiation factor.

Aims

Most electrophysiological studies on Alzheimer's Disease (AD) have been performed using AD mouse models. The electrophysiological properties of neurons in the human AD brain have not been described due to the lack of suitable material for such investigations.

Methods

Here we describe, using the whole-cell patch-clamp technique in acute slices, the electrophysiological properties of human neurons in cortical biopsies obtained from idiopathic normal pressure hydrocephalus patients undergoing shunt surgery. Due to the beta-amyloid and tau pathology present in the subpopulation of these biopsies, they offer a tissue model for investigation on how AD-related pathology affects neuronal operational properties in the human brain.

Results

We recorded several neuronal parameters including sodium and potassium currents, the shape and action potential firing pattern, and spontaneous post-synaptic activity of both pyramidal cells and fast-firing parvalbumin interneurons in these biopsies. We found alterations in the excitation and inhibition balance in neurons in biopsies with AD-related pathology. Morphological reconstruction of the recorded neurons filled with biocytin revealed alterations in the spine density in neurons recorded in biopsies with AD-related pathology.

Conclusions

This is the first study to report the impact of AD-related pathology on single-neuron operational properties and morphology. Our integrative analysis of human neuronal electrophysiology and subsequent cell morphology reconstruction allows us to register primary pathological changes in neuronal functions in correlation with spine distribution impacted by AD pathology.

Aims

Alzheimer’s disease is a synaptic disorder. While the etiology of late-onset Alzheimer’s disease (LOAD) is likely multifactorial, genetic predisposition accelerates the disease onset. CD2AP was identified as a genetic risk factor for LOAD. However, how CD2AP contributes to LOAD synaptic dysfunction is unclear. We have shown that CD2AP loss-of-function increases β-amyloid (Abeta) endocytic production. We now aim to determine if it contributes to synapse dysfunction via Abeta or by playing a role in synapses. Because CD2AP is an actin-binding protein, we hypothesized it might function in F-actin-rich dendritic spines, the excitatory postsynaptic compartment.

To establish if CD2AP, a late Alzheimer’s disease risk factor, impacts synapses via Abeta or F-actin.

Methods

We analyzed wild-type primary mouse cortical neurons knockdown or overexpressing CD2AP and determined the impact of a LOAD mutation in CD2AP function using a sensitive cell biological and neurobiological approach to characterize study synapses, amyloidogenesis and the actin cytoskeleton.

Results

We demonstrate that CD2AP colocalizes with F-actin in dendritic spines. Cell-autonomous depletion of CD2AP specifically reduces spine density and volume, with a functional decrease in synapse formation and neuronal network activity. Postsynaptic reexpression of CD2AP but not blocking Abeta-production is sufficient to rescue spine density. CD2AP overexpression increases spine density, volume, and synapse formation, while a rare LOAD CD2AP mutation induces aberrant F-actin spine-like protrusions without synapses. CD2AP controls postsynaptic actin turnover, with the LOAD mutation in CD2AP decreasing F-actin dynamicity.

Conclusions

Our data support that CD2AP risk variants could contribute to LOAD synapse dysfunction by deregulating actin dynamics, which disrupts spine formation and growth upstream of Abeta production.

Aims

Neuronal pentraxin-2 (NPTX2), crucial for synaptic functioning, declines in cerebrospinal fluid (CSF) as cognition deteriorates. The variations of CSF NPTX2 across mild cognitive impairment (MCI) due to Alzheimer's disease (AD) and its association with brain metabolism remain elusive, albeit relevant for patient stratification and pathophysiological insights.

Methods

We retrospectively analyzed 49 MCI-AD patients grouped by time until dementia (EMCI, n=34 progressing within two years; LMCI, n=15 progressing later/stable at follow-up). We analyzed demographic variables, cognitive status (MMSE score), and CSF NPTX2 levels using a commercial ELISA assay in EMCI, LMCI, and a control group of age-/sex-matched individuals with other non-dementing disorders (OND). Using [¹⁸F]FDG PET scans for voxel-based analysis we explored topographical correlation between brain metabolism and CSF NPTX2 levels in MCI-AD patients, accounting for age.

Results

Baseline and follow-up MMSE scores were lower in LMCI than EMCI (p-value=0.006 and p<0.001). EMCI exhibited significantly higher CSF NPTX2 values than both LMCI (p=0.028) and OND (p=0.006). We found a significant positive correlation between NPTX2 values and metabolism of bilateral precuneus in MCI-AD patients (p<0.005 at voxel level, p<0.05 with FWE correction at the cluster level).

Conclusions

Higher CSF NPTX2 in EMCI compared to controls and LMCI suggests compensatory synaptic response to initial AD pathology. Disease progression sees overwhelmed mechanisms, lowering CSF NPTX2 approaching dementia. Positive CSF NPTX2 correlation with precuneus glucose metabolism links to AD-related metabolic changes across MCI course. These findings posit CSF NPTX2 as a promising biomarker for both AD staging and progression risk stratification

Aims

We have previously used APP23 mice to show that amyloid-β clearance and thereby memory improvements can be achieved by transient blood-brain barrier (BBB) opening upon treatment with intravenously injected microbubbles and 1 MHz scanning ultrasound (SUS^+MB) (Leinenga & Götz, Science Transl Med, 2015). However, given that we had also shown more recently in senescent mice that SUS without microbubbles (SUS^only, no BBB opening) can restore LTP induction and cognition (Blackmore et al., Mol Psych 2021), we here asked whether such a treatment would restore memory in APP23 mice in the absence of amyloid-β clearance.

Methods

The study comprised four treatment arms: untreated wild-type mice, and APP23 mice that received 8 weekly treatments in a SUS^only mode (1 MHz or 286 kHz) or sham. Assignment to treatment groups was done based on matching memory performance (APA test). After the treatments, mice underwent a memory re-test, followed by an MRI analysis and a proteomic, biochemical and histological analysis.

Results

We found that repeated SUS^only treatment at 1 MHz frequency can ameliorate memory deficits in APP23 mice without reducing amyloid-β burden. Different from previous studies that had shown amyloid-β clearance as a consequence of BBB opening, here, the BBB was not opened as no microbubbles were used. Quantitative proteomics and functional magnetic resonance imaging revealed that ultrasound induced long-lasting functional changes that correlate with the improvement in memory. Intriguingly, the treatment was more effective at a higher frequency (1MHz) than at a frequency within the range currently explored in clinical trials in AD patients (286 kHz).

Conclusions

Together, our data suggest frequency-dependent bio-effects of ultrasound and a dissociation of cognitive improvement and amyloid-β clearance, with important implications for the design of trials for AD therapies.

Aims

The amyloid precursor protein (APP) is a key player in Alzheimer`s disease (AD) and the precursor of the Aβ peptide, which is generated by consecutive cleavages of β- and γ-secretases. Familial Alzheimer’s disease (FAD) describes a hereditary subgroup of AD with an early onset of the disease. Different APP FAD mutations are thought to have qualitatively different effects on its proteolytic conversion. However, few studies have explored the pathogenic and putative physiological differences in more detail. Here, we compared different FAD mutations, located at the β- (Swedish), α- (Flemish, Arctic, Iowa) or γ-secretase (Iberian) cleavage sites. We examined heterologous expression of APP WT and FAD mutants in non-neuronal cells and their impact on presynaptic differentiation in contacting axons of co-cultured neurons.

Methods

We tested subcellular localization, endocytosis rate and proteolytic processing by immunoprecipitation–mass spectrometry.

Results

We found that only the Iberian mutation showed altered synaptogenic function. Furthermore, the APP Iowa mutant shows significantly decreased α-secretase processing which is in line with our result that APP carrying the Iowa mutation was significantly increased in early endosomes. However, most interestingly, immunoprecipitation–mass spectrometry analysis revealed that the amino acid substitutions of APP FAD mutants have a decisive impact on their processing reflected in altered Aβ profiles. Importantly, N-terminally truncated Aβ peptides starting at position 5 were detected preferentially for APP Flemish, Arctic, and Iowa mutants containing amino acid substitutions around the α-secretase cleavage site. The strongest change in the ratio of Aβ40/Aβ42 was observed for the Iberian mutation while APP Swedish showed a substantial increase in Aβ1-17 peptides.

Conclusions

Together, our data indicate that familial AD mutations located at the α-, β-, and γ-secretase cleavage sites show considerable differences in the underlying pathogenic mechanisms.