MiNE Nanoparticle Characterization

MiNE NPs are poly(ethylene glycol)-modified (PEGylated), biocompatible liposomes with an aqueous core encapsulating a validated pro-MFN2 peptide (DIAEAVRGIMDSLHMAAR) with a molecular weight of 1956.26 g/mol [21]. The published pro-MFN2 peptide was designed as a cell penetrating TAT-conjugated peptide to bind to amino acids 716–736 of MFN2 as a potential treatment for Charcot Marie Tooth disease type 2 A which is caused by MFN2 mutations [21]. Instead of relying on a TAT sequence to facilitate cell entry, our approach is to use liposome nanoparticles to deliver the pro-MFN2 peptide. Figure 1 illustrates the predicted structure and characterization of the peptide. The pro-MFN2 structure is predicted to be primarily helical with disbursed coils and extended regions. The grand average of hydropathicity (GRAVY) score is 0.461 with predicted cytoplasmic stability and demonstrated aqueous solubility. Due to the properties of the peptide and aqueous compatibility, liposomes were selected as the NP delivery system. Table 1 lists the NP characterization data. Blank NPs (without the peptide) have an average hydrodynamic diameter of 161.4 nm while the average particle size for MiNE NPs is 229.7 nm, respectively. The zeta potential values for both the blank and MiNE NPs indicates particle stability (outside of the + 30 mV to −30 mV range). The peptide encapsulation efficiency of MiNE NPs averaged 76.38%. All drug dosing was according to molarity and adjusted according to batch encapsulation efficiency. MiNE NPs are composed of 44% dipalmitoylphosphatidylcholine (DPPC; an amphipathic phospholipid ideal for bilayer formation), 14% cholesterol for structural integrity, 22% 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP; a cationic lipid to facilitate preferential mitochondrial association), and 20% polyethylene glycol (PEG; a hydrophilic polymer that decreases particle aggregation and decreases immune clearance). Neutral MiNE NPs were also synthesized and evaluated (data not shown). Neutral particles had slow/minimal peptide release and low activity. Cationic MiNE NPs demonstrated rapid peptide release and superior activity and, as mitochondria are the most negatively charged organelle in the cell, cationic NPs most likely aid in mitochondrial localization of the peptide. These NPs are not actively targeting mitochondria but the charge attraction is a form of passive targeting.

Table 1 Liposome characterizationMiNE Nanoparticles Increase Mitochondrial Networks

Mitochondrial Network Analysis (MiNA) software was used to quantify MiNE NP induced changes in mitochondrial networks in primary Sprague Dawley rat hippocampal neurons and in NIH3T3 fibroblasts [50]. The software was developed as a compatible macro to run through NIH Image J [50]. A series of image processing steps are conducted to generate a 3D skeleton of mitochondrial networks [50]. The 3D image is then analyzed to determine nine parameters including the number of individual mitochondria, the number of networks, the network size (number of branches), and the mitochondrial footprint [50]. The mitochondrial footprint accounts for the flaws of the mathematical expression and is a quantification of the binary signal of fluorescence [50]. Our prior studies validated the application of this method [51]. As illustrated in Fig. 2, the 60X objective of a Keyence BZ-X710 microscope was used to acquire brightfield and fluorescent images of primary rat hippocampal neurons stained with Mitotracker™ Green. Figure 2 are each a set of representative images from the four treatment groups. All images captured an area of 257.28 × 192.96 microns. Column 1 is a panel of images from untreated neurons, column 2 is a panel of images from Mine NP treated neurons, column 3 is a panel of images from Blank (no peptide) NP treated neurons, while column 4 is a panel of images from neurons treated with the pro-MFN2 peptide in solution. The rows of images are; (1) the fluorescent overlay on the brightfield channel, (2) the green fluorescent channel which represents mitochondrial staining with Mitotracker™ Green FM, (3) bright field, (4) the binary converted image of the green channel, (5) the skeletonized green fluorescent channel, (6) the skeleton alone, (7) the 2D rendering of the mitochondrial network generated from the skeleton, and (8) the 3D construction of the mitochondrial network based on the mitochondrial network morphology, branching pattern, and mathematical parameters in the MiNA software. The mitochondrial footprint is the total mitochondrial signal and is measured from the binary image (4). All other parameters including the number of individual mitochondria and the number and size of mitochondrial networks are quantified from the 3D network (8). The scale bar on the skeletonized green fluorescent channel (5) and the 3D mitochondrial network (8) is 64 µm. Supplemental data (Supplemental Figs. 1 and 2) are additional neuronal panels of treated and untreated cells.

Fig. 2The alternative text for this image may have been generated using AI.

Microscopy and mitochondrial network analysis of primary hippocampal neurons (Panel 1). Cells were stained with Mitotracker™ Green, fluorescent microscopy was performed using the 60X objective of a Keyence BZ-X710 microscope. Images were processed using mitochondrial network analysis (MiNA) software. Columns represent the different treatments: A Untreated, B MiNE NPs, C Blank NPs, D Pro-MFN2 Peptide in Solution. Rows represent the different image processing: (1) the fluorescent overlay on the brightfield channel, (2) the green fluorescent channel which represents mitochondrial staining with Mitotracker™ Green FM, (3) bright field, (4) the binary converted image of the green channel, (5) the skeletonized green fluorescent channel, (6) the skeleton alone, (7) the 2D rendering of the mitochondrial network generated from the skeleton, and (8) the 3D construction of the mitochondrial network based on the mitochondrial network morphology, branching pattern, and mathematical parameters in the MiNA software

Figure 3 is the quantitative analysis of the mitochondrial imaging of the neurons; Fig. 2 are representative image panels from the total n = 10. As the cells are primary hippocampal neurons an n = 10 was the maximum n due to growth/propagation limitations. The panels in Fig. 2 illustrate the differences between the treatment groups after 2 h of 10 µM treatment, with row 4 (binary) and row 8 (3D network) being the most important parameters that are used to derive the mitochondrial footprint and all network parameters. A two hour time point was selected as our previous studies in cancer cells have demonstrated that this is a long enough time period to induce changes in mitochondrial networks [51].

Fig. 3The alternative text for this image may have been generated using AI.

Mitochondrial Network Analysis (MiNA) Demonstrates that, After Two Hours, MiNE NPs Increase the Number of Mitochondrial Networks in Primary Hippocampal Neurons. MiNA of 10 samples per group was performed to quantify changes induced by treatments. MiNE NPs and to a lesser extent, pro-MFN2 solution, increased the number of individual mitochondria, the number of mitochondrial networks, and the mitochondrial footprint. GraphPad Prism was used to conduct a Welch’s t-test (n = 10) and graph mean with SD. *p < 0.05, ** p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001

Fig. 4The alternative text for this image may have been generated using AI.

Microscopy and mitochondrial network quantification of untreated and MiNE NP Treated NIH3T3 Fibroblasts. Cells were stained with Mitotracker™ Green, fluorescent microscopy was performed using the 60X objective of a Keyence BZ-X710 microscope. Images were processed using mitochondrial network analysis (MiNA). Rows: A Untreated Cells, B MiNE NP Treated Cells, C Blank NPs, D Pro-MFN2 Peptide in Solution. Columns: (1) represents an overlay of the green fluorescent channel with a scalebar of 20 µM (2) and the brightfield image (3). Columns 4–7 are MiNA processing of the stained mitochondrial skeleton (4), binary conversion of the skeleton (5), 2D network analysis (6), and 3D network quantification (7). An n = 20 was used for each group

Panel A of Fig. 3 illustrates that MiNE NPs and the pro-MFN2 peptide solution increase the number of individual mitochondria relative to untreated neurons and neurons treated with the blank (no peptide) NPs. Cells treated with blank NPs were dosed at the same volume of NP to media ratio as the cells treated with MiNE NPs to maintain the same amount of liposomes as a control. Untreated neurons had an average of 216 individual mitochondria per area (257.28 × 192.96 microns), while MiNE NP treated had an average of 488.7, blank NP treated had 256.3, and neurons treated with peptide solution had 451.8. Panel B of Fig. 3 demonstrates that MiNE NPs significantly increase the number of mitochondrial networks relative to all other treatment groups. Blank NPs also increase the number of mitochondrial networks. This may be due to an activating interaction of the PEG or lipid constituents of the NPs with mitochondria and this will be explored further in the future. Although blank NPs increase mitochondrial networks, the pro-MFN2 peptide solution has a greater capacity to increase mitochondrial networks than the blank NPs. The average number of mitochondrial networks per area for the untreated neurons was 12.9, this average increased to 21.1 after treatment with blank NPs, and to 25.9 after treatment with the pro-MFN2 peptide solution. The most significant increase in mitochondrial networks was after treatment with MiNE NPs; the average number of networks per area was 37.5 after 2 h of MiNE NP treatment at 10 µM. This is 1.45 fold higher than peptide solution treatment, 1.78 fold higher than treatment with blank NPs, and 2.9 fold higher than untreated neurons. Although the mitochondrial networks in neurons are sensitive to activation from blank NPs and peptide solution after 2 h of treatment, the activity of the peptide solution is expected to be brief as the peptide in solution is vulnerable to degradation. The current in vitro experiment is in a stationary 2D cell culture experiment; both the blank NPs and peptide solution are expected to have little to transient activity in vivo due to degradation, biodistribution, and non-saturating conditions. The NP component of MiNE NPs is expected to protect the pro-MFN2 peptide and enable cellular uptake after intranasal administration to the brain. Future work will explore the effect of blank NPs on mitochondrial networks which may result in synergistic activity with the peptide.

Even though the treatment groups increase the number of mitochondrial networks, as represented by Panels C and D of Fig. 3, there is no significant increase in the mean branch length of networks and in the mean network size (number of branches). This is most likely due to conformational restrictions of neuron morphology. As neurons have an octopus-like structure with a rounded cell body and thin, spindly axon and dendrite extensions, we hypothesize that there is not enough space within the axons and dendrites to significantly expand the mitochondrial network size and length of branches.

Treatment with MiNE NPs as well as the pro-MFN2 peptide solution significantly increases the mitochondrial footprint with more of a significant increase with the MiNE NP treatment (Panel E, Fig. 3). The footprint is the total mitochondrial signal from the binary image (row 4, Fig. 2). The footprint is measured to compensate for flaws with the MiNA software. The MiNE NP and peptide solution treated neurons have more mitochondrial signal per area (257.28 × 192.96 microns) than the untreated and blank NP treated neurons. An intersection that will be explored in future studies is how mitochondrial networks, mitophagy, and the rate of mitochondrial transfer between neurons relate. Mitophagy is critical to neuronal homeostasis and defective mitophagy has been correlated to the development of AD and PD [52,53,54,55,56]. It is possible that the increase in mitochondrial networks achieved with MiNE NPs increases the rate and/or efficiency of mitophagy which would result in a higher number of healthy mitochondria and the observed increase in mitochondrial footprint. Treatment may also increase the rate of mitochondrial transfer. As observed during this study, after MiNE NP treatment, bi-directional, rapid, real-time mitochondrial transfer was observed between the axon and dendrites of neighboring neurons (Supplemental Data Video 1). Rapid intra-neuron movement of mitochondria was also observed (Supplemental Data Video 2). Future studies will explore this intersection and quantify the kinetics of mitochondrial transfer and the rate of mitophagy.

As a control, mitochondrial networks in NIH3T3 fibroblasts were also quantified in untreated cells, cells treated with 10 µM MiNE NPs for two hours, cells treated with blank NPs for two hours, and cells treated with 10 µM of the pro-MFN2 peptide for two hours (Fig 4). Due to the morphology of these cells it was possible to do single cell analysis, which is ideal because interference can be minimized and the mitochondrial network parameters can be normalized to the total square micron area of the cell. Due to the intertwined morphology of neurons, single cell analysis of neurons was not conducted yet all cells were seeded at the same density. For the single cell fibroblast analysis, an n of 20 was used for each treatment group. Prior to imaging, NIH3T3 cells were stained with Mitotracker™ Green. Panel 4A is a representative untreated cell while Panel 4B is a representative MiNE NP treated cell (10 uM). Panel 4C is a representative cell treated with blank NPs while Panel 4D is a representative cell treated with pro-MFN2 peptide in solution. The columns are (1) the fluorescent overlay on the brightfield channel, (2) the green fluorescent channel, (3) bright field, (4) skeletonized green channel, (5) binary image, (6) 2D network, and (7) 3D network. The mitochondrial footprint is measured from the binary image (5) while all other parameters are quantified from the 3D network (7). Figure 5 illustrates the parameters measured from the 3D network images. Panel 5A demonstrates that MiNE NPs decrease the number of individual mitochondria per square micron relative to untreated cells and cells treated with the pro-MFN2 peptide in solution. Interestingly, unlike MiNE NPs increasing the number of mitochondrial networks in neurons but not increasing the network size, MiNE NPs grow existing networks in fibroblasts without increasing the number of networks. Panel 5B demonstrates that there is no significant change in the number of mitochondrial networks after MiNE NP treatment. MiNE NPs were effective at growing existing mitochondrial networks (Panel 5C). Panel 5A and 5C suggest MiNE NPs are facilitating individual mitochondria adding to existing network branches as the network size represents the number of branches per network. In fibroblasts, MiNE NPs are not changing the total fluorescent signal from mitochondria although the peptide solution slightly increased the mitochondrial footprint.

Fig. 5The alternative text for this image may have been generated using AI.

Mitochondrial Network Analysis (MiNA) Demonstrates that, After Two Hours, MiNE NPs Decrease Individual Mitochondria and Increase Mean Networks Size in NIH3T3 Cells. MiNA of 20 samples per group was performed to quantify changes induced by treatments. The footprint is the entire fluorophore staining per square microns. To account for size differences in cells, all data is normalized to the square micron area of each image. GraphPad Prism was used to conduct a Welch’s t-test (n = 20) and graph mean with SEM

This variation of increasing the number of mitochondrial networks but not the network size in neurons and increasing the size of existing networks but not the number of networks in fibroblasts may be due to the simple difference of cell morphology. Unlike the octopus-like structure of neurons that have narrow and restricted mass in axons and dendrites, fibroblasts are more triangular, globular, and expansive. It is possible that, in fibroblasts, the response to grow mithondrial networks after MiNE NP treatment may be more beneficial than inducing new network formation as there is a higher capacity for expansive, larger networks than in neurons. A future question that may be explored is if there is a threshold where mitochondrial networks reach a maximum size in fibroblasts and, at this threshold, does the response to MiNE NPs shift to the induction of new mitochondrial networks? The ability of MiNE NPs to increase the number of mitochondrial networks in neurons and increase the size of existing mitochondrial networks in fibroblasts is very promising for increasing mitochondrial function. Mitochondria in network conformation are more resistant to cell death signaling and have a higher capacity for producing ATP.

MiNE Nanoparticles Increase Mitochondrial-Endoplasmic Reticulum Co-localization

The function of mitochondria in fusing to the ER is critical for maintaining ER health and rescuing the ER from the unfolded protein response [33, 38,39,40,41]. The ability of MiNE NPs to increase mitochondrial fusion with ER was evaluated in co-localization studies (Figs. 6 and 7). Due to growth restraints of the primary neurons, this study was only conducted in fibroblasts. Cells were stained with Mitotracker™ Green and Bodipy ER Red and fluorescent microscopy was performed using the 60X objective of a Zeiss LSM 880 microscope to obtain images of untreated cells (Fig. 6A) and MiNE NP treated cells (Fig. 6B). The columns represent the (1) overlay of the three channels, (2) bright field, (3) green mitochondrial channel, and (4) red ER channel. Image J and the JACoP plugin (a colocalization macro) were used to process the images and three colocalization statistical values were used to determine the differences between treated and untreated groups: Pearson’s correlation coefficient, Mander’s split coefficients for ER over mitochondria and mitochondria over ER. The first value analyzed, Pearson’s Correlation Coefficient, determines the overlap of fluorescence in each channel without regard for intensity. Pearson’s correlation coefficient is a measure of spatial co-occurrence of fluorescence and output is expressed at values ranging from −1 to 1 with the value of −1 being perfect anticorrelation, 0 being no correlation, and 1 being perfect correlation. The value gives an effective measure of how much area colocalization exists in. The second colocalization analytic utilized is the Mander’s split coefficient, which gives insight into the intensity of fluorescence for each group. Mander’s output measures the fraction of intensity of the desired channel as a value from 0 to 1 in all pixels where the concurrent channel exhibits a non-zero value.

Fig. 6The alternative text for this image may have been generated using AI.

Microscopy of Mitochondria (Green) and ER (Red) Colocalization. A Untreated NIH3T3 Cells. B MiNE NP treated cells. Cells were stained with Mitotracker™ Green and Bodipy ER Red, fluorescent microscopy was performed using the 60X objective of a Zeiss LSM 880 microscope. (1) Overlay of the three channels (yellow signal indicates co-localization), (2) bright field, (3) green mitochondrial channel, (4) red endoplasmic reticulum channel. Representative images of n = 20

Fig. 7The alternative text for this image may have been generated using AI.

Mitochondrial-Endoplasmic Reticulum Co-localization. Colocalization of untreated and MiNE NP treated cells measured through Pearson and Mander’s coefficients for pixel overlap. Data analysis for mitochondrial-ER colocalization was also conducted through ImageJ using the JACoP plugin. GraphPad Prism 9.0 (Mean and SD)

In Fig. 7, all statistical determinants of colocalization show that cells treated with MiNE NPs exhibited higher levels of colocalization compared to control cells. Although the yellow regions of Fig. 6, Panel 1 are a visual representation of the green and red overlap, for the quantitative analysis in Fig. 7, the pixel overlap was measured. Higher levels of colocalization imply mitochondria and the ER occupy the same position in cell. Pearson’s coefficient provides insight to the overall colocalization and shows that MiNE NP treated cells experience larger total overlap of mitochondria and ER. Both Mander’s coefficients show that the intensity of fluorescence is increased in MiNE NP treated cells. This could indicate the possibility that cells are experiencing higher levels of mitochondrial-ER contact. Future studies will evaluate MiNE NP induced mitochondrial-ER co-localization in neurons and include the negative controls of blank NPs, and pro-MFN2 peptide solution.

The accumulation of misfolded proteins and protein aggregates is a hallmark of neurodegenerative disease and a consequence of ER inadequacy. The ability of MiNE NPs to increase mitochondrial co-localization with the ER could increase ER efficiency, decrease the accumulation of misfolded proteins and, as further studies demonstrate, rescue the ER from the UPR.

MiNE Nanoparticles Increase Oxidative Phosphorylation

For decades, school children around the world have been programmed to memorize “mitochondria are the powerhouse of the cell”. The energy producing function of mitochondria are undoubtedly their most famous quality. Mitochondrial dysfunction in aging and neurodegenerative disease is associated with energy depletion [2, 8,9,10,11,12]. The effect of MiNE NPs on OXPHOS activity was assessed (Fig. 8) using Abcam’s MitoTox™ Complex V OXPHOS Activity Assay Kit as Complex V is the final ATP producing subunit of the OXPHOS machinery. The kit uses extracted bovine cardiac mitochondria.

Fig. 8The alternative text for this image may have been generated using AI.

MiNE NPs Significantly Increase Complex V Activity Relative to Normal OXPHOS and Blank NP Treatment. Abcam's MitoTox™ Complex V OXPHOS Assay demonstrated that MiNE NPs and dose escalation increase Complex V activity. MiNE NPs treatments included 1 µM, 10 µM, 25 µM, 50 µM, and 100 µM. PBS was used as a negative control. n = 7. From 1 µM to 50 µM MiNE NPs there was an increase in Complex V activity corresponding to the increased dose. The significance of this increase decreased with the 25 µM to 50 µM transition and seems to plateau between 50 to 100 µM indicating there may be a saturation or other plateau event. Significance was determined using Welch's t-test in GraphPad Prism 9.0 (Mean and SD). *p < 0.05, ** p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001

The effect of increasing (1 µM—100 µM) concentrations of MiNE NPs on purified mitochondria complex v activity were evaluated continuously for 2 hours. The rate of the reaction (change in OD per minute) was determined using the data at 30 min and 120 min and converted to percent activity. As shown in Fig. 8, treatment with PBS, blank NPs, and 1 µM MiNE NPs resulted in 100% complex V activity. Treatment with 10 µM significantly increased complex v activity to an average of 136.5% while treatment with 25 µM significantly increased activity to an average of 154.2%. Although with less significance, treatment with 50 µM significantly increased activity to an average of 176.5% while there was not a significant increase with 100 µM MiNE NP treatment. MiNE NPs demonstrate a sigmodal response in complex v activity. The lack of response for 1 µM is expected to be due to the low dose of the peptide while the lack of increase at 100 µM could represent a saturation of complex v activity or the plateau of MiNE NP efficacy. The ability of MiNE NPs to increase complex v activity in purified mitochondria suggests that mitochondrial fusion/pro-MFN2 activity could directly increase OXPHOS, offering a new treatment avenue for increasing energy capacity in neurodegenerative disease.

MiNE Nanoparticles Protect Against the Unfolded Protein Response

Mitochondria are not the only dysfunctional organelle in neurodegenerative disease. The accumulation of misfolded and aggregated proteins is well documented in neurodegenerative disease which is associated with a level of ER deficiency [30,31,32]. The UPR in neurodegenerative disease is under-investigated, but it has been noted to be increased in AD [44, 45]. There is a close intersection of the UPR, mitochondrial-ER fusion, mitophagy/autophagy, and cell death/survival decision points [33, 38,39,40,41,42]. The ability of MiNE NPs to protect against the UPR was assessed in both the NIH3T3 fibroblasts and in the primary hippocampal neurons (Fig. 9). Thapsigargin (Thaps), a positive control provided with the kit, was used to induce the UPR. The UPR induced by thapsigargin co-incubated with 10 µM and 25 µM MiNE NPs and pro-MFN2 solution was evaluated at time zero and 6 h post treatment, with % change correlating to an increase in the UPR measured via a fluorescent reporter. In both cell lines, the pro-MFN2 solution did not rescue cells from thapsigargin induced UPR. Over the course of 6 h, MiNE NPs (both at 10 µM and 25 µM) significantly decreased the thapsigargin induced UPR. Interestingly, this effect was more significant in the primary hippocampal neurons than in the immortalized NIH3T3 fibroblasts. There is a plethora of possibilities for this, spanning from cellular sensitivity to the UPR to differences in mitochondrial-ER ratios. In both cell lines, blank NPs had no effect on preventing the UPR. Mitochondrial fusion to the ER has been reported to rescue the ER from the UPR [33, 38,39,40,41]. The ability of MiNE NPs to decrease the ER response to stress and prevent the induction of the UPR could slow the detrimental effects of misfolded protein accumulation in neurodegenerative disease.

Fig. 9The alternative text for this image may have been generated using AI.

MiNE NPs Protect Against the Unfolded Protein Response. The Montana Molecular UPR Assay was used to quantify ER stress and was compared to the known UPR inducer thapsigargin (Thaps). Activity was measured over 6 hours of treatment and cell stressor in NIH3T3 cells (left 6 columns)) and in primary hippocampal neurons (right 6 most columns). Significance is only shown for MiNE NPs. There was no significance between thapsigargin and all other treatments. Significance was determined using Welch's t-test in GraphPad Prism 9.0. n = 9 with triplicate studies (Mean and SD). *p < 0.05, ** p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001

MiNE Nanoparticles Potential Mechanism of Action

These preliminary studies of MiNE NPs have demonstrated that MiNE NPs can increase the number of mitochondrial networks in neurons, grow mitochondrial networks in fibroblasts, increase mitochondrial-ER co-localization in fibroblasts, increase complex v activity in purified bovine cardiac mitochondria, and protect fibroblasts and neurons from the ER stress induced unfolded protein response. As mitochondrial dysfunction and misfolded protein accumulation are central to neurodegenerative disease, MiNE NPs have strong potential as a future treatment for AD and PD. Future studies will further explore the mechanisms of action and biological effects of MiNE NPs. As illustrated in Fig. 10, we further hypothesize that MiNE NPs could increase apoptotic resistance through the conformationally protective orientation of networked mitochondria which decreases pro-apoptotic Bcl2 family member accessibility to binding sites on the OMM. By increasing mitochondrial health (through increased efficiency of OXPHOS functioning and increased mitophagy), a decrease in ROS accumulation is expected to result in a decrease in mtDNA damage and cellular ROS induced damage. Through increased mitochondrial fusion with the ER, MiNE NPs are expected to increase protein synthesis and folding efficiency (under non-UPR and UPR conditions). This outcome would be transformative in the treatment of AD and PD. Through the UPR axis, MiNE NPs could increase mitophagy and autophagy, increasing mitochondrial health and overall cellular survival. As in the videos of inter-neuron mitochondrial transfer and intra-neuron movement, (Supplemental Data Videos 1 and 2), future studies will also evaluate if MiNE NPs increase mitochondrial movement and transfer between cells. MiNE NPs are the first treatment for neurodegenerative disease to manipulate mitochondria and the ER. This organelle level approach could calm the perfect storm of mitochondrial dysfunction that contributes to the development and progression of neurological disease.

Fig. 10The alternative text for this image may have been generated using AI.

Mitochondrial Network Enhancing (MiNE) Nanoparticle Hypothesized Mechanism of Action. MiNE NPs increase mitochondrial network number or size (A) and this study demonstrated that this increase in network number or size results in (1) increased energy capacity as demonstrated by Complex V activity. We also hypothesize (and will test in the future), that this increased mitochondrial network size will (2) increase apoptotic resistance as networked mitochondria are conformationally protective against pro-apoptotic signaling. Increased network number or size is also hypothesized to (3) decrease ROS due to increased mitochondrial health through the fusion- mitophagy axis. MiNE NPs also (B) increase mitochondrial to endoplas