Small-molecule G-quadruplex stabilizers reveal a novel pathway of autophagy regulation in neurons

International audienc

SPHK1/sphingosine kinase 1-mediated autophagy differs between neurons and SH-SY5Y neuroblastoma cells

Although implicated in neurodegeneration, autophagy has been characterized mostly in yeast and mammalian non-neuronal cells. In a recent study, we sought to determine if SPHK1 (sphingosine kinase 1), implicated previously in macroautophagy/autophagy in cancer cells, regulates autophagy in neurons. SPHK1 synthesizes sphingosine-1-phosphate (S1P), a bioactive lipid involved in cell survival. In our study, we discovered that, when neuronal autophagy is pharmacologically stimulated, SPHK1 relocalizes to the endocytic and autophagic organelles. Interestingly, in non-neuronal cells stimulated with growth factors, SPHK1 translocates to the plasma membrane, where it phosphorylates sphingosine to produce S1P. Whether SPHK1 also binds to the endocytic and autophagic organelles in non-neuronal cells upon induction of autophagy has not been demonstrated. Here, we determined if the effect in neurons is operant in the SH-SY5Y neuroblastoma cell line. In both non-differentiated and differentiated SH-SY5Y cells, a short incubation of cells in amino acid-free medium stimulated the formation of SPHK1-positive puncta, as in neurons. We also found that, unlike neurons in which these puncta represent endosomes, autophagosomes, and amphisomes, in SH-SY5Y cells SPHK1 is bound only to the endosomes. In addition, a dominant negative form of SPHK1 was very toxic to SH-SY5Y cells, but cultured primary cortical neurons tolerated it significantly better. These results suggest that autophagy in neurons is regulated by mechanisms that differ, at least in part, from those in SH-SY5Y cells

Levetiracetam mitigates doxorubicin-induced DNA and synaptic damage in neurons

Neurotoxicity may occur in cancer patients and survivors during or after chemotherapy. Cognitive deficits associated with neurotoxicity can be subtle or disabling and frequently include disturbances in memory, attention, executive function and processing speed. Searching for pathways altered by anti-cancer treatments in cultured primary neurons, we discovered that doxorubicin, a commonly used anti-neoplastic drug, significantly decreased neuronal survival. The drug promoted the formation of DNA double-strand breaks in primary neurons and reduced synaptic and neurite density. Pretreatment of neurons with levetiracetam, an FDA-approved anti-epileptic drug, enhanced survival of chemotherapy drug-treated neurons, reduced doxorubicin-induced formation of DNA double-strand breaks, and mitigated synaptic and neurite loss. Thus, levetiracetam might be part of a valuable new approach for mitigating synaptic damage and, perhaps, for treating cognitive disturbances in cancer patients and survivors

Cytoplasmic sphingosine-1-phosphate pathway modulates neuronal autophagy

Autophagy is an important homeostatic mechanism that eliminates long-lived proteins, protein aggregates and damaged organelles. Its dysregulation is involved in many neurodegenerative disorders. Autophagy is therefore a promising target for blunting neurodegeneration. We searched for novel autophagic pathways in primary neurons and identified the cytosolic sphingosine-1-phosphate (S1P) pathway as a regulator of neuronal autophagy. S1P, a bioactive lipid generated by sphingosine kinase 1 (SK1) in the cytoplasm, is implicated in cell survival. We found that SK1 enhances flux through autophagy and that S1P-metabolizing enzymes decrease this flux. When autophagy is stimulated, SK1 relocalizes to endosomes/autophagosomes in neurons. Expression of a dominant-negative form of SK1 inhibits autophagosome synthesis. In a neuron model of Huntington's disease, pharmacologically inhibiting S1P-lyase protected neurons from mutant huntingtin-induced neurotoxicity. These results identify the S1P pathway as a novel regulator of neuronal autophagy and provide a new target for developing therapies for neurodegenerative disorders

Regulation of autophagy by DNA G-quadruplexes

International audienceGuanine-rich DNA strands can form secondary structures known as G-quadruplexes (G4-DNA). G4-DNA is important for the regulation of replication and transcription. We recently showed that the expression of Atg7, a gene that is critical for macroautophagy/autophagy, is controlled by G4-DNA in neurons. We demonstrated that the transcription factor SUB1/PC4 and the G4-DNA-specific antibody HF2 bind to a putative G4-DNA motif located in the Atg7 gene. Stabilizing G4-DNA with the G4-ligand pyridostatin (PDS) downregulates Atg7 expression in neurons. Here, we further investigated how G4-DNA in the Atg7 gene is stabilized by PDS. We show that PDS can form 1:1 and 2:1 complexes with the Atg7's G4. We also demonstrate that PDS downregulates the ATG7 protein and the expression of Atg7 in astrocytes as well as in neurons. Together with our previous findings, these data establish a novel G4-DNA-associated mechanism of autophagy regulation at a transcriptional level in neurons and astrocytes

Differential responses of neurons, astrocytes, and microglia to G-quadruplex stabilization

International audienceThe G-quadruplex (G4-DNA or G4) is a secondary DNA structure formed by DNA sequences containing multiple runs of guanines. While it is now firmly established that stabilized G4s lead to enhanced genomic instability in cancer cells, whether and how G4s contribute to genomic instability in brain cells is still not clear. We previously showed that, in cultured primary neurons, small-molecule G4 stabilizers promote formation of DNA double-strand breaks (DSBs) and downregulate the Brca1 gene. Here, we determined if G4-dependent Brca1 downregulation is unique to neurons or if the effects in neurons also occur in astrocytes and microglia. We show that primary neurons, astrocytes and microglia basally exhibit different G4 landscapes. Stabilizing G4-DNA with the G4 ligand pyridostatin (PDS) differentially modifies chromatin structure in these cell types. Intriguingly, PDS promotes DNA DSBs in neurons, astrocytes and microglial cells, but fails to downregulate Brca1 in astrocytes and microglia, indicating differences in DNA damage and repair pathways between brain cell types. Taken together, our findings suggest that stabilized G4-DNA contribute to genomic instability in the brain and may represent a novel senescence pathway in brain aging