Abstract Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer’s Disease (AD). While neurons generally produce a minority of the apoE in the central nervous system, neuronal expression of apoE increases dramatically in response to stress and is sufficient to drive pathology. Currently, the molecular mechanisms of how apoE4 expression may regulate pathology are not fully understood. Here we expand upon our previous studies measuring the impact of apoE4 on protein abundance to include the analysis of protein phosphorylation and ubiquitylation signaling in isogenic Neuro-2a cells expressing apoE3 or apoE4. ApoE4 expression resulted in a dramatic increase in VASP S235 phosphorylation in a PKA-dependent manner. This phosphorylation disrupted VASP interactions with numerous actin cytoskeletal and microtubular proteins. Reduction of VASP S235 phosphorylation via PKA inhibition resulted in a significant increase in filopodia formation and neurite outgrowth in apoE4-expressing cells, exceeding levels observed in apoE3-expressing cells. Our results highlight the pronounced and diverse impact of apoE4 on multiple modes of protein regulation and identify protein targets to restore apoE4-related cytoskeletal defects.
Abstract The PTEN-induced putative kinase 1 knockout rat (Pink1−/−) is marketed as an established model for Parkinson’s disease, characterized by development of motor deficits and progressive degeneration of half the dopaminergic neurons in the substantia nigra pars compacta by 8 months of age. In this study, we address our concerns about the reproducibility of the Pink1−/− rat model. We evaluated behavioural function, number of substantia nigra dopaminergic neurons and extracellular striatal dopamine concentrations by in vivo microdialysis. Strikingly, we and others failed to observe any loss of dopaminergic neurons in 8-month-old male Pink1−/− rats. To understand this variability, we compared key experimental parameters from the different studies and provide explanations for contradictory findings. Although Pink1−/− rats developed behavioural deficits, these could not be attributed to nigrostriatal degeneration as there was no loss of dopaminergic neurons in the substantia nigra and no changes in neurotransmitter levels in the striatum. To maximize the benefit of Parkinson’s disease research and limit the unnecessary use of laboratory animals, it is essential that the research community is aware of the limits of this animal model. Additional research is needed to identify reasons for inconsistency between Pink1−/− rat colonies and why degeneration in the substantia nigra is not consistent.
Abstract Background Multiple AD risk genes are implicated in lipid metabolism, and plasma and brain lipid levels are altered in AD. Astrocytes are enriched in key lipid‐related factors and are likely contributors to altered lipid homeostasis in AD. We hypothesize that APP/Aβ‐related pathology and neuroimmune factors modulate astrocytic gene transcription that promote maladaptive changes in lipid pathways, including aberrant astrocytic production and release of lipids that could affect Aβ pathology and neuronal deficits. Method To investigate the effects of APP/Aβ and neuroinflammation on astrocytic lipid metabolism, we used homozygous mutant APP knock‐in mice (APP SAA ‐KI) that have progressive Aβ proteinopathy and gliosis, as well as mice injected systemically with lipopolysaccharide (LPS) to induce acute neuroinflammatory responses. Astrocyte‐specific analyses were performed in vivo by either FACS‐sorting astrocytes for downstream lipidomic profiling or by ribosome‐tagging astrocytes in mouse models (Ribotag) for astrocyte‐specific RNA sequencing. We also performed lipidomic, RNA, and protein‐based analyses in primary murine astrocytes stimulated with oligomeric Aβ (oAβ) or interleukin‐1 (IL‐1α/β). Result Lipidomics in acutely sorted cortical astrocytes from APP SAA ‐KI or LPS‐injected mice revealed decreased levels of triglycerides and cholesterol esters. In LPS‐injected mice, cortical astrocytes also had increased expression of enzymes involved in lipolysis, most notably adipose triglyceride lipase ( Atgl ), the principal triglyceride hydrolase. Similarly, primary astrocytes had increased levels of triglyceride lipolysis upon IL‐1α/β or oAβ stimulation, leading to increased liberation of free fatty acids (FFAs) in an NF‐κB and ERK1/2‐dependent manner. Astrocytic FFAs generated from triglyceride lipolysis were not used for energy production via β‐oxidation but instead utilized by astrocytes to produce prostaglandins (PGE 2 ) and to facilitate secretion of extracellular vesicular bodies (EVB). Inhibiting ATGL decreased PGE 2 production, revealing an essential role of astrocyte triglyceride lipolysis in neuroinflammatory processes. Conclusion Our findings suggest that Aβ pathology and neuroinflammatory factors cause astrocytic triglyceride lipolysis, a process that liberates fatty acids to be utilized for various potentially pathogenic cascades in disease. We conclude that amyloid pathology and neuroimmune factors converge to alter astrocytic lipid‐related gene transcription and lipid‐based signaling.
Abstract Increased α-synuclein (αsyn) and mitochondrial dysfunction play central roles in the pathogenesis of Parkinson’s disease (PD), and lowering αsyn is under intensive investigation as a therapeutic strategy for PD. Increased αsyn levels disrupt mitochondria and impair respiration, while reduced αsyn protects against mitochondrial toxins, suggesting that interactions between αsyn and mitochondria influences the pathologic and physiologic functions of αsyn. However, we do not know if αsyn affects normal mitochondrial function or if lowering αsyn levels impacts bioenergetic function, especially at the nerve terminal where αsyn is enriched. To determine if αsyn is required for normal mitochondrial function in neurons, we comprehensively evaluated how lowering αsyn affects mitochondrial function. We found that αsyn knockout (KO) does not affect the respiration of cultured hippocampal neurons or cortical and dopaminergic synaptosomes, and that neither loss of αsyn nor all three (α, β and γ) syn isoforms decreased mitochondria-derived ATP levels at the synapse. Similarly, neither αsyn KO nor knockdown altered the capacity of synaptic mitochondria to meet the energy requirements of synaptic vesicle cycling or influenced the localization of mitochondria to dopamine (DA) synapses in vivo . Finally, αsyn KO did not affect overall energy metabolism in mice assessed with a Comprehensive Lab Animal Monitoring System. These studies suggest either that αsyn has little or no significant physiological effect on mitochondrial bioenergetic function, or that any such functions are fully compensated for when lost. These results implicate that αsyn levels can be reduced in neurons without impairing (or improving) mitochondrial bioenergetics or distribution.
