Aims

Calcium stores and store operated calcium (SOC) entry channels have emerged recently as viable early triggers of AD. Mutated presenilin1 (mPS1) is assumed to underlie one of the major routes of neuronal deterioration occurring in familial AD patients. While extensive work attempts to connect PS1 to regulation of intracellular calcium concentration ([Ca²⁺]i), the exact mechanism affected by mPS1 to cause [Ca²⁺]i dysregulation remains unclear.

Abstract Body

Mutated presenilin1 (mPS1) is assumed to underlie one of the major routes of neuronal deterioration occurring in familial AD patients. Using imaging and electrophysiological methodologies, we examined the role of PS1 in regulation of calcium stores associated with endoplasmic reticulum and mitochondria in cultured rat hippocampal neurons. We transfected cultured rat hippocampal neurons with PS1 as well as the mutated PS1-M146V plasmids, along with one of several marker proteins to detect cell morphology, mitochondrial calcium concentration ([Ca²⁺]m), endoplasmic reticulum (ER) calcium, or mitochondrial motility. Free intracellular calcium ([Ca²⁺]i) was estimated with the calcium sensor Fluo-2AM. Dendritic and spine morphology of mPS1 were different from those of PS1 transfected neurons. Strikingly, replenishment of [Ca²⁺]i after its depletion by removal of extracellular calcium, caused a fast and marked increase in both [Ca²⁺]i and [Ca²⁺]m. However, the change in [Ca²⁺]m was smaller and slower in neurons transfected with mPS1 than in controls. The functional significance of the presence of mPS1 was demonstrated in the responses of the transfected neuron to paraquat (PQ), an oxidative stressor. Exposure of the neurons to PQ increased mitochondrial motility in mPS1, but not in PS1 neurons. Strikingly, 24hrs exposure to PQ caused death of neurons, much more among the mPS1 cells than among controls. These experiments highlight the link between oxidative stress, and the balance in calcium regulation between the endoplasmic reticulum and the mitochondria. These results corroborate and extend observations on the role of calcium regulation in mitochondria and ER in the aged animals.

Methods

We transfected cultured rat hippocampal neurons with PS1 as well as the mutated PS1-M146V plasmids, along with one of several marker proteins to detect cell morphology, mitochondrial calcium concentration ([Ca²⁺]m), endoplasmic reticulum (ER) calcium, or mitochondrial motility. Free intracellular calcium ([Ca²⁺]i) was estimated with the calcium sensor Fluo-2AM.

Results

Replenishment of [Ca²⁺]i after its depletion by removal of extracellular calcium, caused a fast and marked increase in both [Ca²⁺]i and [Ca²⁺]m. However, the change in [Ca²⁺]m was smaller and slower in neurons transfected with mPS1 than in controls. The responses of the transfected neuron to paraquat (PQ), an oxidative stressor were measured. Exposure of the cultured neurons to PQ caused a significant increase in rate of network bursts in mPS1 cells, but not in the PS1 cells. Also, PQ increased mitochondrial motility in mPS1, but not in PS1 transfected neurons. Finally, 24hrs exposure to PQ caused death of neurons, much more among the mPS1 cells than among controls.

Conclusions

These experiments highlight the link between oxidative stress, and the balance in calcium regulation between the endoplasmic reticulum and the mitochondria.

Abstract Body

Sigma-1 receptor (S1R) is an endoplasmic reticulum resident transmembrane protein and changes in its expression or sequence are associated with neurodegenerative phenotypes. There are several S1R agonists that are developed for treating neurodegenerative disorders, some of them are in human clinical trials. However, biological function of S1R in neurons is poorly understood. To get insights into S1R function in cells we studied S1R localization to mitochondrial-associated membrane (MAM) domains and biophysical determinants of S1R localization. We identified protein motifs responsible for S1R-cholesterol interactions that are essential for proper S1R targeting to the MAM regions. We demonstrated formation of S1R clusters in the presence of cholesterol in giant unilamellar vesicles (GUV) reconstitution model system. We propose that S1R functions as a lipid raft-stabilizing protein of the ER that is critical for formation and function of MAMs and other ER contact sites.

In experiments with hippocampal neuronal cultures from PS1-KI and APP-KI mouse models of AD we demonstrated that S1R agonists 3-PPP and pridopidine prevented the loss of mushroom spines in these cultures. Rescue effects of Pridopidine were abolished when S1R expression levels were suppressed by siRNA. In experiments with hippocampal slices pridopidine reversed LTP defects resulting from application of Aβ42 oligomers. Daily treatment of PS1-KI-GFP mice 30 mg/kg pridopidine daily prevented hippocampal mushroom spine loss in vivo. These results suggested that ligands of S1R can potentially expert a beneficial effect in AD and that this function most likely related to remodeling of MAMs and other ER contact sites in neurons.

Aims

Amyloid-beta oligomers (AβOs) are synaptotoxins that induce aberrant Ca²⁺-signals and promote ROS generation, leading to synaptic plasticity disruption. We have reported that AβOs induce a sustained but low-intensity cytoplasmic [Ca²⁺]i increase in neurons, which arises from NMDA receptor-mediated Ca²⁺ entry and subsequent amplification via Ca²⁺-induced Ca²⁺-release, mediated by ryanodine receptor (RyR) channels. We also reported that RyR2 isoform has a key role in the mitochondrial dysfunctions induced by AβOs-treatment in neurons, and in the memory-defects observed in rats injected with AβOs. Since gene expression changes are mediated by the rapid activation and expression of transcription factors, we evaluated the expression of RyR2 and distinct activity-dependent genes regulating the expression of neurotrophic factors and antioxidant enzymes in response to AβOs. We also tested the combined effects of AβOs and Gabazine (GBZ), a GABA(A)-receptor blocker that functions as an inductor of synaptic activity.

Methods

Primary hippocampal cultures were pre-incubated for 6 h with AβOs before the Gabazine (GBZ) stimulation for 2h. RyR2, Npas4, BDNF, Glutamate-Cysteine-Ligase (GCL) and NADPH-Quinone-Oxidoreductase (Nqo1) mRNA expression levels were determined by RT-qPCR.

Results

GBZ alone induced in just 2 h an increase in RyR2 expression, BDNF, Npas4, glutamate-Cysteine-Ligase (GCL), and NADPH-Quinone-Oxidoreductase (Nqo1). Conversely, pre-treatment with AβOs prevented these increases in the mRNA expression levels of these proteins induced by GBZ.

Conclusions

We propose that AβOs block the activation of signaling pathways induced by stimulation of neuronal activity by GBZ, leading to a disruption of the neuroprotective gene expression pathways essential to memory and learning processes, which are affected in neurodegenerative diseases.

Abstract Body

We describe mechanistically-distinct enzymes, i.e., a ubiquitin protein hydrolase (Uch-L1), a kinase (Cdk5) and a guanosine triphosphatase (Drp1), which function in disparate biochemical pathways, that can also act in concert to mediate a series of redox reactions that contribute to neurodegenerative disorders. We show that each enzyme manifests a second, non-canonical function – transnitrosylation – triggering a pathological biochemical cascade in Alzheimer’s disease (AD). In this chemical redox reaction, NO (most likely in the form of nitrosonium cation or NO⁺) reacts with cysteine thiol (or more properly thiolate anion, R-S^-). The S-nitrosylation reaction mechanism involves thiolate anion, as a nucleophile, performing a reversible nucleophilic attack on the nitroso nitrogen to form an SNO-protein adduct. The resulting series of kinetically and thermodynamically-favored transnitrosylation reactions contributes to synapse loss, the major pathological correlate to cognitive decline in AD. Moreover, we develop a quantitative method based on a series of Nernst equations for thermodynamic assessment of the reactions at steady state, as might be expected to occur in a chronic disease. This analysis revealed Gibbs free energies that predict the spontaneous forward reaction of the transnitrosylation cascade. While other potential members of the cascade remain to be determined, this work shows that non-canonical pathways mediating a concerted cascade of aberrant transnitrosylation reactions can contribute to the pathogenesis of neurodegenerative disorders. We conclude that enzymes with distinct primary reaction mechanisms can form a completely separate network for aberrant transnitrosylation. This network operates in the post-reproductive period, so natural selection against such abnormal activity may be decreased.

Aims

A number of studies on AD patients and mouse models have reported aberrant neuronal activity early in the course of the disease. This suggests that synaptic functions are out of balance, but it remains enigmatic how accumulation of amyloid-β (Aβ) in plaques and hyperphosphorylated Tau in tangles interferes with this process. Alterations in neuronal activity recruit compensatory mechanisms to renormalize synapse function and to maintain circuit stability. However, long-term alterations can lead to the failure of these mechanisms, triggering neuronal malfunction and initiating the transition into cognitive impairment.

Methods

By combining electrophysiological recordings with single-cell and spatial transcriptomic profiling we set-out to dissect the molecular signatures of synapse renormalization and maintenance of circuit stability in the humanized knock-in mouse for mutated amyloid precursor protein (APP^NL-G-F), when the first biochemical insults appear.

Results

We found that in the APP^NL-G-F mice but also in AD patients, a sleep-dependent process involved in synapse renormalization is impaired.

Conclusions

These findings pave the way for new therapeutic interventions to stabilize network activity.

Aims

Astrocytes generate spontaneous calcium events whilst performing homeostatic functions essential for optimal cerebral function. We hypothesized that spontaneous astrocytic calcium activity is hyperactive throughout cellular compartments within the awake APP/PS1 mouse brain.

Methods

We expressed the ratiometric calcium indicator, yellow cameleon, specifically within cortical astrocytes of 12-17 months old APP/PS1 transgenic mice (n=10) and non-transgenic littermates (n=9). Using multiphoton microscopy, we qualitatively and quantitatively measured spontaneous astrocytic calcium activity, through a chronic craniotomy over the somatosensory cortex, of awake habituated mice. Time-lapse imaging was analyzed using custom-written MATLAB scripts.

Results

‘Silent’ astrocytes, with a stable YFP/CFP ratio, accounted for 17/60 (28%) astrocytes in wild-type mice and 22/69 (32%) astrocytes in APP/PS1 mice. There were 12 spontaneous ‘localized’ events within the astrocytic primary processes of wild-type mice compared to 25 ‘localized’ events within the astrocytic primary processes of APP/PS1 mice. Wild-type astrocytic endfeet exhibited 27 ‘localized’ events compared to 11 ‘localized’ events within the astrocytic endfeet of APP/PS1 mice. ‘Multicompartmental’ events occurred with greater amplitude in the somas of APP/PS1 mice when compared to wild-type mice (p=0.009). Heterogeneous ‘multicellular’ events occurred with greater amplitude in the somas (p=0.01) and primary processes (p=0.0029) of APP/PS1 mice when compared to wild-type mice.

Conclusions

Here we reveal the heterogeneous complexity, compartmentalization and pathological hyperactivity of spontaneous intra- and inter-cellular calcium activity, most likely coupled to aberrant astrocytic function, within the astrocytic network and neurovascular unit of the awake APP/PS1 mouse brain. These data suggest that therapeutics targeting astrocyte activity represent an alternative target for treating Alzheimer’s disease.

Aims

Dysregulation of the Endoplasmic Reticulum (ER) Calcium homeostasis^1-4, and subsequent ER stress activation occur in Alzheimer’s disease (AD). The human truncated isoform of the Sarco-endoplasmic reticulum Calcium ATPase 1 (S1T)⁵ triggers and amplifies ER stress response, leading to subsequent cell commitment to apoptosis through the control of Calcium mobilization from ER to mitochondria⁶. We examined S1T expression in AD and investigated the mutual link between S1T expression and APP processing and neuroinflammation.

Methods

We used SH-SY5Y cells overproducing beta-amyloid precursor protein-derived fragments (APPswe) or treated with oligomeric Amyloid beta (Abeta) peptides, 3xTg-AD transgenic mice and human brains. We used biochemical, quantitative RT-PCR, and sterotaxic injection in 3xTg-AD mice brains.

Results

S1T expression is increased in SH-SY5Y cells expressing APPswe or treated with Abeta oligomers and in sporadic AD brains and is correlated with Abeta load and key ER stress proteins. Overexpression of S1T enhances in return the production of APP C-terminal fragments and Abeta through specific increases of beta-secretase expression and activity. Elevated S1T expression also triggers neuroinflammation in vitro and in vivo⁷.

Conclusions

We describe a molecular interplay between S1T-dependent ER Calcium leak, ER stress and APP processing that could contribute to AD pathogenesis.

1-Oules B,et al. 2012, J Neurosci.

2-Del Prete D, et al. 2014, Mol Neurodegener .

3-Lacampagne A, et al. 2017, Acta Neuropathol.

4-Bussiere R, et al. 2017, J Biol Chem .

5-Chami M, et al. 2001, J Cell Biol.

6-Chami M, et al. 2008, Mol Cell.

7-Bussiere R, et al. 2019, Cells.

Aims

CD2-Associated protein (CD2AP) is associated with an increased risk of late-onset Alzheimer’s disease (AD). We previously show that loss of CD2AP’s Drosophila homolog, cindr, exacerbates Tau toxicity and impairs synaptic maturation and short-term plasticity in flies. However, little is understood about CD2AP’s role at the central mammalian synapse. The present study investigates CD2AP’s role at the post-synapse and in long-term plasticity.

Methods

Using a CD2AP knockout mouse (CD2AP^-/-), we examined short- and long-term plasticity via electrophysiological recordings and assessed morphological changes in primary neuronal cultures. Since CD2AP^-/- mice die within 6-7 weeks of age, CD2AP heterozygous mice were also examined.

Results

CD2AP is expressed ubiquitously in the brain and enriched at the mouse hippocampus. Using hippocampal slice electrophysiology, we show that both heterozygous and homozygous CD2AP knockouts mice exhibit enhanced paired-pulse facilitation when compared to controls. Although tetanic stimulation performed in young (4-6 weeks old) mice shows inductions of long-term potentiation (LTP) similar to that seen in controls, we observed decreased LTP in aged CD2AP heterozygous mice. In cell culture, CD2AP^-/- neurons exhibit increased dendritic branching and PSD95 expression when compared to controls suggesting that CD2AP regulates post-synaptic structure and function.

Conclusions

Taken together, our study shows conserved function of CD2AP for short-term plasticity in flies and mice and suggest haploinsufficient requirements of CD2AP for LTP in an age-dependent manner. Ongoing research will further characterize CD2AP at the post-synapse and in a tauopathy model of AD.