APP-dependent glial cell line-derived neurotrophic factor gene expression drives neuromuscular junction formation.

Besides its crucial role in the pathogenesis of Alzheimer's disease, the knowledge of amyloid precursor protein (APP) physiologic functions remains surprisingly scarce. Here, we show that APP regulates the transcription of the glial cell line-derived neurotrophic factor (GDNF). APP-dependent regulation of GDNF expression affects muscle strength, muscular trophy, and both neuronal and muscular differentiation fundamental for neuromuscular junction (NMJ) maturation in vivo In a nerve-muscle coculture model set up to modelize NMJ formation in vitro, silencing of muscular APP induces a 30% decrease in secreted GDNF levels and a 40% decrease in the total number of NMJs together with a significant reduction in the density of acetylcholine vesicles at the presynaptic site and in neuronal maturation. These defects are rescued by GDNF expression in muscle cells in the conditions where muscular APP has been previously silenced. Expression of GDNF in muscles of amyloid precursor protein null mice corrected the aberrant synaptic morphology of NMJs. Our findings highlight for the first time that APP-dependent GDNF expression drives the process of NMJ formation, providing new insights into the link between APP gene regulatory network and physiologic functions.-Stanga, S., Zanou, N., Audouard, E., Tasiaux, B., Contino, S., Vandermeulen, G., René, F., Loeffler, J.-P., Clotman, F., Gailly, P., Dewachter, I., Octave, J.-N., Kienlen-Campard, P. APP-dependent glial cell line-derived neurotrophic factor gene expression drives neuromuscular junction formation.

An evaluation of the self-assembly enhancing properties of cell-derived hexameric amyloid-ß.

A key hallmark of Alzheimer's disease is the extracellular deposition of amyloid plaques composed primarily of the amyloidogenic amyloid-ß (Aß) peptide. The Aß peptide is a product of sequential cleavage of the Amyloid Precursor Protein, the first step of which gives rise to a C-terminal Fragment (C99). Cleavage of C99 by γ-secretase activity releases Aß of several lengths and the Aß42 isoform in particular has been identified as being neurotoxic. The misfolding of Aß leads to subsequent amyloid fibril formation by nucleated polymerisation. This requires an initial and critical nucleus for self-assembly. Here, we identify and characterise the composition and self-assembly properties of cell-derived hexameric Aß42 and show its assembly enhancing properties which are dependent on the Aß monomer availability. Identification of nucleating assemblies that contribute to self-assembly in this way may serve as therapeutic targets to prevent the formation of toxic oligomers.

Mechanism of Cellular Formation and In Vivo Seeding Effects of Hexameric ß-Amyloid Assemblies.

The ß-amyloid peptide (Aß) is found as amyloid fibrils in senile plaques, a typical hallmark of Alzheimer's disease (AD). However, intermediate soluble oligomers of Aß are now recognized as initiators of the pathogenic cascade leading to AD. Studies using recombinant Aß have shown that hexameric Aß in particular acts as a critical nucleus for Aß self-assembly. We recently isolated hexameric Aß assemblies from a cellular model, and demonstrated their ability to enhance Aß aggregation in vitro. Here, we report the presence of similar hexameric-like Aß assemblies across several cellular models, including neuronal-like cell lines. In order to better understand how they are produced in a cellular context, we investigated the role of presenilin-1 (PS1) and presenilin-2 (PS2) in their formation. PS1 and PS2 are the catalytic subunits of the γ-secretase complex that generates Aß. Using CRISPR-Cas9 to knockdown each of the two presenilins in neuronal-like cell lines, we observed a direct link between the PS2-dependent processing pathway and the release of hexameric-like Aß assemblies in extracellular vesicles. Further, we assessed the contribution of hexameric Aß to the development of amyloid pathology. We report the early presence of hexameric-like Aß assemblies in both transgenic mice brains exhibiting human Aß pathology and in the cerebrospinal fluid of AD patients, suggesting hexameric Aß as a potential early AD biomarker. Finally, cell-derived hexameric Aß was found to seed other human Aß forms, resulting in the aggravation of amyloid deposition in vivo and neuronal toxicity in vitro.

Presenilin-Deficient Neurons and Astrocytes Display Normal Mitochondrial Phenotypes.

Presenilin 1 (PS1) and Presenilin 2 (PS2) are predominantly known as the catalytic subunits of the γ-secretase complex that generates the amyloid-ß (Aß) peptide, the major constituent of the senile plaques found in the brain of Alzheimer's disease (AD) patients. Apart from their role in γ-secretase activity, a growing number of cellular functions have been recently attributed to PSs. Notably, PSs were found to be enriched in mitochondria-associated membranes (MAMs) where mitochondria and endoplasmic reticulum (ER) interact. PS2 was more specifically reported to regulate calcium shuttling between these two organelles by controlling the formation of functional MAMs. We have previously demonstrated in mouse embryonic fibroblasts (MEF) an altered mitochondrial morphology along with reduced mitochondrial respiration and increased glycolysis in PS2-deficient cells (PS2KO). This phenotype was restored by the stable re-expression of human PS2. Still, all these results were obtained in immortalized cells, and one bottom-line question is to know whether these observations hold true in central nervous system (CNS) cells. To that end, we carried out primary cultures of PS1 knockdown (KD), PS2KO, and PS1KD/PS2KO (PSdKO) neurons and astrocytes. They were obtained from the same litter by crossing PS2 heterozygous; PS1 floxed (PS2+/-; PS1flox/flox) animals. Genetic downregulation of PS1 was achieved by lentiviral expression of the Cre recombinase in primary cultures. Strikingly, we did not observe any mitochondrial phenotype in PS1KD, PS2KO, or PSdKO primary cultures in basal conditions. Mitochondrial respiration and membrane potential were similar in all models, as were the glycolytic flux and NAD+/NADH ratio. Likewise, mitochondrial morphology and content was unaltered by PS expression. We further investigated the differences between results we obtained here in primary nerve cells and those previously reported in MEF cell lines by analyzing PS2KO primary fibroblasts. We found no mitochondrial dysfunction in this model, in line with observations in PS2KO primary neurons and astrocytes. Together, our results indicate that the mitochondrial phenotype observed in immortalized PS2-deficient cell lines cannot be extrapolated to primary neurons, astrocytes, and even to primary fibroblasts. The PS-dependent mitochondrial phenotype reported so far might therefore be the consequence of a cell immortalization process and should be critically reconsidered regarding its relevance to AD.

Amyloid Precursor Protein (APP) Controls the Expression of the Transcriptional Activator Neuronal PAS Domain Protein 4 (NPAS4) and Synaptic GABA Release.

The amyloid precursor protein (APP) has been extensively studied as the precursor of the ß-amyloid (Aß) peptide, the major component of the senile plaques found in the brain of Alzheimer's disease (AD) patients. However, the function of APP per se in neuronal physiology remains to be fully elucidated. APP is expressed at high levels in the brain. It resembles a cell adhesion molecule or a membrane receptor, suggesting that its function relies on cell-cell interaction and/or activation of intracellular signaling pathways. In this respect, the APP intracellular domain (AICD) was reported to act as a transcriptional regulator. Here, we used a transcriptome-based approach to identify the genes transcriptionally regulated by APP in the rodent embryonic cortex and on maturation of primary cortical neurons. Surprisingly, the overall transcriptional changes were subtle, but a more detailed analysis pointed to genes clustered in neuronal-activity dependent pathways. In particular, we observed a decreased transcription of neuronal PAS domain protein 4 (NPAS4) in APP-/- neurons. NPAS4 is an inducible transcription factor (ITF) regulated by neuronal depolarization. The downregulation of NPAS4 co-occurs with an increased production of the inhibitory neurotransmitter GABA and a reduced expression of the GABAA receptors α1. CRISPR-Cas-mediated silencing of NPAS4 in neurons led to similar observations. Patch-clamp investigation did not reveal any functional decrease of GABAA receptors activity, but long-term potentiation (LTP) measurement supported an increased GABA component in synaptic transmission of APP-/- mice. Together, NPAS4 appears to be a downstream target involved in APP-dependent regulation of inhibitory synaptic transmission.

Presenilin 2-Dependent Maintenance of Mitochondrial Oxidative Capacity and Morphology.

Mitochondrial dysfunction plays a pivotal role in the progression of Alzheimer's disease (AD), and yet the mechanisms underlying the impairment of mitochondrial function in AD remain elusive. Recent evidence suggested a role for Presenilins (PS1 or PS2) in mitochondrial function. Mutations of PSs, the catalytic subunits of the γ-secretase complex, are responsible for the majority of inherited AD cases (FAD). PSs were shown to be present in mitochondria and particularly enriched in mitochondria-associated membranes (MAM), where PS2 is involved in the calcium shuttling between mitochondria and the endoplasmic reticulum (ER). We investigated the precise contribution of PS1 and PS2 to the bioenergetics of the cell and to mitochondrial morphology in cell lines derived from wild type (PS+/+), PS1/2 double knock-out (PSdKO), PS2KO and PS1KO embryos. Our results showed a significant impairment in the respiratory capacity of PSdKO and PS2KO cells with reduction of basal oxygen consumption, oxygen utilization dedicated to ATP production and spare respiratory capacity. In line with these functional defects, we found a decrease in the expression of subunits responsible for mitochondrial oxidative phosphorylation (OXPHOS) associated with an altered morphology of the mitochondrial cristae. This OXPHOS disruption was accompanied by a reduction of the NAD+/NADH ratio. Still, neither ADP/ATP ratio nor mitochondrial membrane potential (ΔΨ) were affected, suggesting the existence of a compensatory mechanism for energetic balance. We observed indeed an increase in glycolytic flux in PSdKO and PS2KO cells. All these effects were truly dependent on PS2 since its stable re-expression in a PS2KO background led to a complete restoration of the parameters impaired in the absence of PS2. Our data clearly demonstrate here the crucial role of PS2 in mitochondrial function and cellular bioenergetics, pointing toward its peculiar role in the formation and integrity of the electron transport chain.