synaptosomes are enriched in neuron-specific and depleted in glia-specific proteins
4.8 Potential improvements and future applications of FASS
positive synaptosomes were also positive for Ly6H. Together, with the enrichment of Ly6H protein in synaptic plasma membrane fractions these results hint at a possible localization of Ly6H to VGLUT1 synapses. However, the results obtained so far are confounded by the lack of specificity of the Ly6H antibody and more specific antibodies for Ly6H will be necessary to validate the localization of Ly6H to VGLUT1 synapses.
In the present study, I show that subpopulations of synaptosomes can be isolated for subsequent biochemical analysis using FASS when VGLUT1VENUS mice are used as a
source of fluorescent synaptosomes. This novel protocol provides an important methodological advance with many possible applications in neuroscience research. In addition this new methodological approach allowed me to address several biological questions and to identify new synaptic proteins. Further improvements of various aspects of the method as well as expected technological advancements will further enhance the usefulness of the FASS methodology and its applications.
4.8.1 Potential for improvements of the current FASS-method
Freshly prepared synaptosomes can be considered as live particles, whose quality deteriorates with longer durations of experiments. The subcellular fractionation prior to FASS requires roughly 3 h in the current protocol, and from one brain one can obtain mg quantities of synaptosomal protein. However, the following sorting of VGLUT1VENUS
Since in the flow-cytometric analysis the non-fluorescent and the fluorescent particles are not completely separated, improvements in the sensitivity of the fluorescence detection system could increase the fraction of VGLUT1
synaptosomes only yields roughly 0.8- 1 µg of synaptosomal protein with sort times of 6-8 h. I observed that during longer sorts the sample quality decreased. Thus, throughput of the cell-sorter is a major factor that limits the amounts of sorted synaptosomes that can be isolated during a single FASS experiment.
VENUS
Furthermore, recent experiments performed by Etienne Herzog on a different sort platform in Paris suggest that the establishment of custom procedures for laser alignment and drop-delay during instrument set-up may contribute to increase the yield during microparticle analysis and sorting.
positive synaptosomes available for FASS. The maximum analysis rate of the instrument during sorting is relatively fixed.
Therefore, increasing the fraction of positive particles will increase the frequency by which particles can be sorted and will thus reduce the total time of the procedure. Potential technical modifications that would allow for a more sensitive fluorescence detection would be the optimization of fluorescence filter sets, an excitation optimized for VENUS at 514 nm and as well as the selection of the optimal PMT (photo-multiplier-tube) used for detection of different wavelengths.
An additional way to improve the protein yield of protein per experiment would be to optimize the sample recovery after FASS. Filtration on 0.22 µm polycarbonate filters recovers 56 % of the initial protein. By using different methods, such as adsorption to a column carrying a high affinity antibody or a lectin with affinity to synaptosomes or alternative methods of protein precipitation could be explored in order to achieve higher recovery rates.
The preparation of synaptosomes using sucrose as a gradient material has been shown to yield optimal results regarding the purify of the synaptosomal preparation
(Whittaker, 1993). However, the use of non-viscous gradient materials such as Percoll
might significantly reduce the time required for sample preparation and would thus reduce the overall time required for the procedure (Dunkley et al., 1986).
Another point for improvement of FASS would be to increase the purity of the sorted VGLUT1VENUS synaptosomes to a level of more than 99 %, as is commonly achieved in the isolation of cells using FACS. The two limiting factors in this respect are the sensitivity and resolution of the light-scattering detection systems on the FACSAria and the sensitivity of fluorescence detection. Improvement of the resolution and sensitivity of the light-scattering detection would allow for proper doublet-discrimination using pulse-geometry by plotting FSC-H vs. FSC-A and SSC-H vs. SSC-A and could thus help to avoid the sorting of coinciding particles. Currently the FACSAria does not have any resolution in the size range of particles that I was isolating. Therefore attempts of introducing doublet discrimination into our sort strategy did not have any effect on sort purity (data not shown). A different sort platform that has recently been developed implements detection of microparticles with more sophisticated light-scatter detection systems and has been reported to resolve particles as small as 0.1 µ This would be very helpful for the optimization of the FASS protocol.
4.8.2 Potential applications of FASS
In the present study, I developed a method to purify VGLUT1VENUS synaptosomes by FASS, which yields synaptosomes of a purity that substantially exceeds the purity of conventionally prepared synaptosomes. Furthermore, the selective isolation of VGLUT1VENUS containing particles allowed for the specific enrichment of VGLUT1 synapses over other types of synapses and extrasynaptic neuronal contaminations present in starting synaptosomal preparation. I also demonstrated that FASS samples can be analyzed by Western blotting and proteomic techniques to investigate differential protein expression between samples.
As outlined in the introduction, major brain disorders such as Alzheimer’s disease or Huntington’s disease affect protein expression or localization at excitatory synapses.
Therefore, FASS of VGLUT1VENUS synaptosomes might be a valuable tool in the investigation of VGLUT1-synapse specific alterations in protein expression or localization in the context of mouse models of these brain disorders.
Furthermore, specific presynaptic marker proteins for other neurotransmitter systems could be labeled in a similar way as VGLUT1VENUS and thus allow the isolation of synaptosomes of a variety of neurotransmitter-phenotypes using FASS. Comparative
proteomic analyses of different synaptosome subpopulations isolated by FASS would allow to screen for neurotransmitter-system specific differences in synaptic protein composition.
One can envision that in the future FASS samples can also be used for functional assays. Since VGLUT1VENUS synaptosomes isolated by FASS are largely devoid of contaminations by glial membranes, FASS may eventually allow to address unresolved scientific questions such as the plasma membrane glutamate uptake at presynapses. Direct uptake of glutamate by presynapses has been suggested since many years (for review see (Danbolt, 2001)), but the studies on the presynaptic uptake of glutamate have always been blurred by the massive expression of GLT1 and GLAST on astrocytic processes. However significant improvements of sample yield will be necessary to allow such experiments.
In a longer-term perspective, one of the great potentials of FASS lies in the ability of flow cytometry to perform multiparametric phenotyping of each analyzed particle separately.
The separation of synapses into neurotransmitter phenotypes may therefore only represent the beginning. In the future, a combination of spectrally separable fluorescently labeled genetic markers, with surface labeling of subtype specific proteins such as neurotransmitter-receptor subunits may allow for the definition and isolation of very specific synaptosome subpopulations using FASS.