Abstract
While mitochondria maintain essential cellular functions, such as energy production, calcium homeostasis, and regulating programmed cellular death, they also play a major role in pathophysiology of many neurological disorders. Furthermore, several neurodegenerative diseases are closely linked with synaptic damage and synaptic mitochondrial dysfunction. Unfortunately, the ability to assess mitochondrial dysfunction and the efficacy of mitochondrial-targeted therapies in experimental models of neurodegenerative disease and CNS injury is limited by current mitochondrial isolation techniques. Density gradient ultracentrifugation (UC) is currently the only technique that can separate synaptic and non-synaptic mitochondrial sub-populations, though small brain regions cannot be assayed due to low mitochondrial yield. To address this limitation, we used fractionated mitochondrial magnetic separation (FMMS), employing magnetic anti-Tom22 antibodies, to develop a novel strategy for isolation of functional synaptic and non-synaptic mitochondria from mouse cortex and hippocampus without the usage of UC. We compared the yield and functionality of mitochondria derived using FMMS to those derived by UC. FMMS produced 3x more synaptic mitochondrial protein yield compared to UC from the same amount of tissue, a mouse hippocampus. FMMS also has increased sensitivity, compared to UC separation, to measure decreased mitochondrial respiration, demonstrated in a paradigm of mild closed head injury. Taken together, FMMS enables improved brain-derived mitochondrial yield for mitochondrial assessments and better detection of mitochondrial impairment in CNS injury and neurodegenerative disease.
Highlights
Mitochondria are small (0.5 to 2 μm) organelles that provide a majority of the cells’ energy in the form of adenosine triphosphate (ATP)
To provide an exact ratio of buffer to brain tissue homogenized and to optimize using our own mitochondrial isolation buffer, we titrated in this recommended range, as to not overload the magnetic column, and tested yield and mitochondrial respiration
We found that a concentration of 10 mL buffer per 75 mg of tissue was optimal for both outcome measures, which is within their recommendation
Summary
Mitochondria are small (0.5 to 2 μm) organelles that provide a majority of the cells’ energy in the form of adenosine triphosphate (ATP). This technique requires a Percoll gradient step (at least 18,000 g spin16) to purify the non-synaptic, or “free,” mitochondrial fraction[18] This technique has been utilized to isolate non-synaptic mitochondria from small regions of the mouse brain, demonstrating relative high yield and purity[16]. While these reports using Percoll gradients do not include disruption of synaptoneurosomes and further isolation of synaptic mitochondria, our laboratory has used this technique to isolate both synaptic and non-synaptic mitochondrial populations[9,19], though requiring higher speed centrifugation (30,400 g spin). These Percoll preparations result in phase bands of mitochondria rather than distinct mitochondrial pellets
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
