Biology subjects

Stanga, S.

Publications and source records attributed to Stanga, S..

5 recordsLinked to original sources

bioRxiv2026

Alzheimer's disease-associated Presenilin 2 N141I mutation impairs neuronal lipid homeostasis and mitochondrial dynamics through selective downregulation of the Golgi exchange factor Gbf1

Presenilin 2 (PS2) mutations cause familial Alzheimers disease, yet their effects beyond amyloid processing remain poorly understood. Here, we investigated how PS2 deletion and the N141I mutation affect neuronal lipid homeostasis and mitochondrial dynamics in mouse primary neurons. Both PS2 deletion and N141I mutation reduced neuronal lipid content. However, exogenous lipid supplementation rescued this deficit only in N141I-expressing neurons, indicating a partial loss-of-function effect. N141I neurons also displayed reduced OPA1, a mitochondrial fusion regulator, restored by lipid supplementation. RNA-sequencing identified Gbf1, a Golgi-specific guanine nucleotide exchange factor, as selectively downregulated in N141I but not knockout tissue, which was confirmed at the protein level in mouse brain and primary neurons. Gbf1 knockdown in mouse embryonic fibroblasts (MEFs) recapitulated the N141I lipid profile. Together, these findings reveal a PS2-GBF1-lipid-mitochondria axis disrupted specifically by the N141I mutation, suggesting an amyloid-independent pathway contributing to neurodegeneration and identifying potential therapeutic targets for familial Alzheimers disease.

neuroscience↗

bioRxiv2021

BBB damage in aging causes brain iron deposits via astrocyte-neuron crosstalk and Hepc/Fpn1 pathway

During aging, iron accumulates in brains regions vulnerable to neurodegeneration: the cerebral cortex and the hippocampus. However, the mechanism of iron regulation in the brain remains scarce. Here, we demonstrated for the first time the involvement of the Hepcidin/Ferroportin1 pathway in brain iron metabolism during aging. We demonstrated the alteration of BBB integrity, that leads to increased iron permeability and deregulation of iron homeostasis during aging. We found that brain iron overload drives Hepcidin upregulation and, consequently, the inhibition of the iron exporter Ferroportin1, neuroinflammation and oxidative stress. Moreover, both in the cerebral cortex and hippocampus Ferroportin1 colocalizes with astrocytes, while the iron storage protein ferritin light-chain with neurons. This differential distribution suggests that astrocytes mediate iron shuttling and neurons are unable to metabolize it. Furthermore, we observed NCOA4-dependent ferritinophagy of ferritin heavy-chain isoforms determining the increase of light-chain enriched ferritin heteropolymers that are more efficient as iron chelators. Altogether, these data highlight the involvement of the Hepcidin/Ferroportin1 axis and NCOA4 during mice aging as a response to a higher iron influx to the brain.

neuroscience↗

bioRxiv2020

NT-mini, a recombinant tool for the study of Neurotrypsin functionality

Neurotrypsin (NT) is a highly specific nervous system multi-domain serine-protease best known for its selective processing of the potent synaptic organiser agrin. Its enzymatic activity is thought to influence processes of synaptic plasticity, with its deregulation causing accelerated neuromuscular junction (NMJ) degeneration or contributing to forms of mental retardation. Something which, based on the available literature, likely stems from NT-based regulation of agrin signalling. However, dissecting the exact biological implications of NT-agrin interplay is difficult, owing to the scarce molecular detail regarding NT activity and NT-agrin interactions. The difficult recombinant production of NT in its catalytically competent form is at the base of these limitations, and is currently constraining a more detailed molecular, biochemical and structural characterisation of the NT-agrin system. We have developed a novel strategy to reliably produce and purify a truncated but catalytically competent variant of NT called NT-mini. The characterisation of our construct highlighted almost wild type-like behaviour with comparable specificity, and conservation of modulation by calcium and heparin despite the lack of several accessory domains. With the data obtained from NT-mini it was then possible to identify NTs heparin-binding domain, and discover a novel putative Zinc-based modulation of NT. Additionally, NT-mini allowed us to investigate the effect of NT activity on myotube formation in controlled cell-based experiments, evidencing a negative impact on myoblast fusion dependant on enzymatic activity. Collectively, this shows the viability of NT-mini as a model to study NT functionality, allowing to expand both in-vitro and "in-cellulo" investigations and providing a foundation to unravel the molecular underpinnings and biological significance of NT-agrin interactions.

biochemistry↗

bioRxiv2020

Presenilin-deficient neurons and astrocytes display normal mitochondrial phenotypes

Presenilins 1 and 2 (PS1 and PS2) are predominantly known as the catalytic subunits of the {gamma}-secretase complex which generates the amyloid-{beta} (A{beta}) peptide, the major constituent of the senile plaques found in the brain of Alzheimers disease (AD) patients. Apart from their role in {gamma}-secretase activity, a growing number of cellular functions have been recently attributed to PSs. They are involved in synaptic transmission, endo-lysosomal function and calcium homeostasis. PSs were also 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 the ER and mitochondria by controlling the formation of functional MAMs through its interaction with the Mitofusin2 protein. We have previously demonstrated that the absence of PS2 (PS2KO) alters mitochondrial morphology and function. Indeed, a PS2KO cell line showed reduced mitochondrial respiration along with disrupted mitochondrial cristae and increased glycolysis. This phenotype is restored by the stable re-expression of human PS2. Still, all these results were obtained in immortalized Mouse Embryonic Fibroblasts (MEF) and one bottom-line question is to know whether these observations hold true for the Central Nervous System (CNS) cells, and in particular neurons and astrocytes. To that end, we carried out primary PS1KO, PS2KO and PS1/PS2KO (PSdKO) neuronal and astrocyte cultures. All the conditions were obtained in the same litter by crossing PS2 heterozygous and PS1 floxed (PS2+/-; PS1flox/flox) animals. Indeed, contrary to PS2KO mice, PS1KO are not viable and therefore require the use of the Cre-LoxP system to achieve gene deletion in vitro. Strikingly, we did not observe any mitochondrial phenotype in PS1KO, PS2KO or PSdKO primary cultures. Mitochondrial respiration and membrane potential were similar in all models, as were the glycolytic flux and NAD+/NADH ratio. We further investigated the discrepancies between these results and the ones previously reported in the MEF PS2KO cell line by analyzing PS2KO primary fibroblasts. No mitochondrial dysfunction was observed in this model, in line with observations in PS2KO primary neurons and astrocytes. These 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 be the consequence of a cell immortalization process and, therefore, should be critically reconsidered regarding its relevance to AD.

neuroscience↗

bioRxiv2018

Amyloid Precursor Protein (APP) controls excitatory/inhibitory synaptic inputs by regulating the transcriptional activator Neuronal PAS Domain Protein 4 (NPAS4)

Sequential proteolysis of the amyloid precursor protein (APP) and amyloid-{beta} peptide (A{beta}) release is an upstream event in Alzheimers disease (AD) pathogenesis. The function of APP in neuronal physiology is still, however, poorly understood. Along with its paralog APP-like Proteins 1 and 2 (APLP1-2), APP is involved in neurite formation and synaptic function by mechanisms that are not elucidated. APP is a single-pass transmembrane protein expressed at high levels in the brain that resembles a cell adhesion molecule or a membrane receptor, suggesting that its function relies on cell interaction processes and/or activation of intracellular pathways of signal transduction. Along this line, the APP intracellular domain (AICD) was reported to act as a transcriptional factor for targeted gene activation that mediates physiological APP functions. Here, we used an unbiased transcriptome-based approach to identify the genes transcriptionally regulated by APP in the rodent embryonic cortex and upon maturation of primary cortical neurons. The transcriptome analysis did not detect any significant differences in expression of previously proposed AICD target genes. The overall transcriptional changes were subtle, but we found that genes clustered in neuronal-activity dependent pathways are dysregulated in the absence of APP. Among these genes, we found the activity-dependent Neuronal PAS domain protein 4 (NPAS4) Immediate Early Gene to be downregulated in the absence of APP. Down-regulation of NPAS4 in APP knock-out (KO) neurons is not related to AICD but to the APP ectodomain. We studied the effect of APP deficiency on GABAergic and glutamatergic transmission, and found an increased production of the inhibitory neurotransmitter GABA in APP KO neurons, along with a reduced expression of the GABA(A) receptors alpha1, suggesting an impaired GABAergic neurotransmission in the absence of APP. CRISPR-Cas-mediated silencing of NPAS4 in neurons led to similar observations. Altogether, our results point out a new role for APP in the regulation of excitatory/inhibitory neurotransmission through the regulation of the activity-dependent NPAS4 gene.

neuroscience↗