Aging Lowers PEX5 Levels in Cortical Neurons in Male and Female Mouse Brains

Peroxisomes are small organelles with critical functions: lipid synthesis, breakdown of reactive oxygen species by antioxidant enzymes, and amino acid degradation. In the brain, peroxisomal lipids make up the myelin sheath. Brain peroxisomal dysfunction leads to lipid disruption or neurological consequences if key peroxisomal proteins are absent. Still, it is unclear how peroxisomes are affected in neurodegenerative diseases and in normal brain aging. This work examines peroxisomal markers in three settings: 1) in a neuronal and 2) animal model of Huntington disease (HD), where mutant huntingtin (mHtt), the causative protein in Huntington disease pathogenesis is expressed, and 3) in the cortices of aged mouse brains. First, we found that the rate of peroxisomes being moved to acidic lysosomes increased in a neuronal HD model compared to control neurons, indicating that mHtt expression amplified the peroxisomal degradation process. We also found that in the cortices of a presymptomatic HD mouse model, neuronal levels of PEX5, (a peroxisomal protein involved in clearance and homeostasis of peroxisomes) and ACAA1 (a peroxisomal marker), were lower than in control groups, suggesting that before HD symptoms begin, mHtt expression may reduce peroxisomal number in neurons, and affect peroxisomal homeostasis by reducing PEX5 levels. These changes, while unexpected, had us wondering if brain aging affected PEX5 levels, since metabolic pathways become impaired as the brain ages. Secondly, we investigated how age and sex affect cortical PEX5 levels of aged male and female mice. We discovered that PEX5 is lower in aged male brains than in young male brains, lower Pex5 cortical expression in aged males compared to younger males, and lower neuronal PEX5 levels in aged male and female cortices, compared to young male and female cortices. In conclusion, aging has a negative effect on neuronal PEX5 levels in aging mouse brains of both sexes. This novel work investigates how PEX5 levels are affected in models of a neurodegenerative disease and in the typical aged mouse brain, setting a foundation for further investigation of the role of peroxisomal proteins in the progression of normal neuronal aging and neurodegenerative disease

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