Synaptoneurosome micromethod for fractionation of mouse and human brain, and primary neuronal cultures

Chang, Julia W.; Arnold, Monica M.; Rozenbaum, Anna; Caputo, Anna; Schweizer, Felix E.; Huynh, My N.; Mathern, Gary W.; Sarafian, Theodore A.; Watson, Joseph B.

doi:10.1016/j.jneumeth.2012.09.005

Cited by 16 publications

(19 citation statements)

References 24 publications

(44 reference statements)

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“…However, a recent report suggests that these truncated forms could also correspond to alternatively spliced forms of human α-synuclein in the ASOTg mice [58]. It is curious that both resected human cortex and PD-related postmortem midbrain homogenates (this study) as well as multiple human cortex SN fractions in our previous study [59] did not show detectable truncation. Possibly truncation of α-synuclein is concentration dependent and is not selective to PD as suggested by a recent report [60].…”

Section: Discussionmentioning

confidence: 67%

“…Resected cortex (central operculum) was obtained from a surgical patient (5.8 years old, female with mild neuronal disorganization, ILAE Cortical Dysplasia I) [65], who underwent neurosurgery at the UCLA’s Pediatric Epilepsy Surgery Program [59]. The research protocols were approved by the Institutional Review Board of the Human Protection Research Committee at the University of California, Los Angeles (UCLA).…”

Section: Methodsmentioning

confidence: 99%

“…Synaptoneurosome Fractionation: Synaptoneurosome (SN) fractions were isolated from freshly dissected mice half-forebrains (50–200 mg wet weight) in a modified Krebs-Henseleit (mKRBS) buffer pH 7.4 [118.5 mM NaCl, 10 mM Dextrose, 4.7 mM KCl, 1.18 mM KH 2 PO 4 , 1.18 mM MgSO 4 , 24.9 mM NaHCO 3 , 2.5 mM CaCl 2, adenosine deaminase (10 µg/ml), benzamide (5 mM), protease inhibitors (leupeptin,1 mg/ml; pepstatin A, 0.005 mg/ml; aprotinin, 0.1 mg/ml), protein phosphatase inhibitors (Na 4 P 2 O 7 , 2 mM; Na 2 MoO 4 , 0.08 mM)] as described previously [11], [36], [59], [67]. SNs were stored at –80°C prior to use in experiments.…”

Section: Methodsmentioning

confidence: 99%

“…Pre-frozen human brain samples were homogenized in mKRBS buffer and filtered once through a nylon mesh as described [59]. Samples were sonicated (3×20 sec) in non-denaturing, non-reducing loading buffer [0.25 M Tris-HCl pH 6.8, 10% (v/v) glycerol, bromophenol blue], centrifuged (1000 x g, 4 min) to remove insoluble material, and loaded without boiling directly onto gels (5% or 10% stacking, 12% resolving).…”

Section: Methodsmentioning

confidence: 99%

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Impairment of Mitochondria in Adult Mouse Brain Overexpressing Predominantly Full-Length, N-Terminally Acetylated Human α-Synuclein

et al. 2013

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While most forms of Parkinson’s Disease (PD) are sporadic in nature, a small percentage of PD have genetic causes as first described for dominant, single base pair changes as well as duplication and triplication in the α-synuclein gene. The α-synuclein gene encodes a 140 amino acid residue protein that interacts with a variety of organelles including synaptic vesicles, lysosomes, endoplasmic reticulum/Golgi vesicles and, reported more recently, mitochondria. Here we examined the structural and functional interactions of human α-synuclein with brain mitochondria obtained from an early, pre-manifest mouse model for PD over-expressing human α-synuclein (ASOTg). The membrane potential in ASOTg brain mitochondria was decreased relative to wildtype (WT) mitochondria, while reactive oxygen species (ROS) were elevated in ASOTg brain mitochondria. No selective interaction of human α-synuclein with mitochondrial electron transport complexes cI-cV was detected. Monomeric human α-synuclein plus carboxyl terminally truncated forms were the predominant isoforms detected in ASOTg brain mitochondria by 2-dimensional PAGE (Native/SDS) and immunoblotting. Oligomers or fibrils were not detected with amyloid conformational antibodies. Mass spectrometry of human α-synuclein in both ASOTg brain mitochondria and homogenates from surgically resected human cortex demonstrated that the protein was full-length and postranslationally modified by N-terminal acetylation. Overall the study showed that accumulation of full-length, N-terminally acetylated human α-synuclein was sufficient to disrupt brain mitochondrial function in adult mice.

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Section: Discussionmentioning

confidence: 67%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Impairment of Mitochondria in Adult Mouse Brain Overexpressing Predominantly Full-Length, N-Terminally Acetylated Human α-Synuclein

et al. 2013

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“…Rat primary hippocampal neurons were isolated according to the protocol described earlier (Chang et al, 2012). Cover glasses coated with poly-L-Lysine (0.1% (v/v) in PBS) were used for seeding isolated mitochondria, HEK293T cells or primary neurons.…”

Section: Methodsmentioning

confidence: 99%

MaxiK channel interactome reveals its interaction with GABA transporter 3 and heat shock protein 60 in the mammalian brain

Singh

Hall

et al. 2016

Neuroscience

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Large conductance voltage and calcium-activated potassium (MaxiK) channels are activated by membrane depolarization and elevated cytosolic Ca2+. In the brain, they localize to neurons and astrocytes, where they play roles such as resetting the membrane potential during an action potential, neurotransmitter release, and neurovascular coupling. MaxiK channels are known to associate with several modulatory proteins and accessory subunits, and each of these interactions can have distinct physiological consequences. To uncover new players in MaxiK channel brain physiology, we applied a directed proteomic approach and obtained MaxiK channel pore-forming α subunit brain interactome using specific antibodies. Controls included immunoprecipitations with rabbit IgG and with anti-MaxiK antibodies in wild type and MaxiK channel knockout mice (Kcnma1−/−), respectively. We have found known and unreported interactive partners that localize to the plasma membrane, extracellular space, cytosol and intracellular organelles including mitochondria, nucleus, endoplasmic reticulum and Golgi apparatus. Localization of MaxiK channel to mitochondria was further confirmed using purified brain mitochondria colabeled with MitoTracker. Independent proof of MaxiK channel interaction with previously unidentified partners is given for GABA transporter 3 (GAT3) and heat shock protein 60 (HSP60). In HEK293T cells, both GAT3 and HSP60 coimmunoprecipitated and colocalized with MaxiK channel; colabeling was observed mainly at the cell periphery with GAT3 and intracellularly with HSP60 with protein proximity indices of ~0.6 and ~0.4, respectively. In rat primary hippocampal neurons, colocalization index was identical for GAT3 (~0.6) and slightly higher for HSP60 (~0.5) association with MaxiK channel. The results of this study provide a complete interactome of MaxiK channel the mouse brain, further establish the localization of MaxiK channel in the mouse brain mitochondria and demonstrate the interaction of MaxiK channel with GAT3 and HSP60 in neurons. The interaction of MaxiK channel with GAT3 opens the possibility of a role of MaxiK channel in GABA homeostasis and signaling.

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Stimulation of synaptoneurosome glutamate release by monomeric and fibrillated α‐synuclein

Sarafian

Littlejohn

Yuan

et al. 2017

J of Neuroscience Research

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The α-synuclein protein exists in vivo in a variety of covalently modified and aggregated forms associated with Parkinson’s disease (PD) pathology. However, the specific proteoform structures involved with neuropathological disease mechanisms are not clearly defined. Since α-synuclein plays a role in presynaptic neurotransmitter release, an in vitro enzyme-based assay was developed to measure glutamate release from mouse forebrain synaptoneurosomes (SNs) enriched in synaptic endings. Glutamate measurements utilizing SNs from various mouse genotypes (WT, over-expressers, knock-outs) suggested a concentration dependence of α-synuclein on calcium/depolarization-dependent presynaptic glutamate release from forebrain terminals. In vitro reconstitution experiments with recombinant human α-synuclein proteoforms including monomers and aggregated forms (fibrils, oligomers) produced further evidence of this functional impact. Notably, brief exogenous applications of fibrillated forms of α-synuclein enhanced SN glutamate release but monomeric forms did not, suggesting preferential membrane penetration and toxicity by the aggregated forms. However, when applied to brain tissue sections just prior to homogenization, both monomeric and fibrillated forms stimulated glutamate release. Immuno-gold and transmission electron microscopy (TEM) detected exogenous fibrillated α-synuclein associated with numerous SN membranous structures including synaptic terminals. Western blots and immuno-gold TEM were consistent with SN internalization of α-synuclein. Additional studies revealed no evidence of gross disruption of SN membrane integrity or glutamate transporter function by exogenous α-synuclein. Overall excitotoxicity, due to enhanced glutamate release in the face of either overexpressed monomeric α-synuclein or extrasynaptic exposure to fibrillated α-synuclein, should be considered as a potential neuropathological pathway during the progression of PD and other synucleinopathies.

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Synaptoneurosome micromethod for fractionation of mouse and human brain, and primary neuronal cultures

Cited by 16 publications

References 24 publications

Impairment of Mitochondria in Adult Mouse Brain Overexpressing Predominantly Full-Length, N-Terminally Acetylated Human α-Synuclein

Impairment of Mitochondria in Adult Mouse Brain Overexpressing Predominantly Full-Length, N-Terminally Acetylated Human α-Synuclein

MaxiK channel interactome reveals its interaction with GABA transporter 3 and heat shock protein 60 in the mammalian brain

Stimulation of synaptoneurosome glutamate release by monomeric and fibrillated α‐synuclein

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