- Drp1 is widely distributed in the central nervous system
To explore the specific distribution of Drp1 in the mouse CNS (central nervous system), we observed its expression in different mouse brain regions and the spinal cord (Fig. 1.A).
To preliminarily explore a potential regional distribution of Drp1 protein, western blot was used to detect the protein in the cortex, hippocampal region, cerebellum, medulla, thalamus and spinal cord. The results showed differences in Drp1 at the protein level in six regions, with the highest expression in the spinal cord and lowest expression in the cortex, hippocampal region and cerebellum (p<0.05) (Fig. 2).
We next explored the exact expression of Drp1 utilizing immunofluorescence staining. Drp1 expression was determined based on the labeling intensity, which was classified as no signal (-), low signal (+), moderate signal (++), high signal (+++), and strong signal (++++)[20].
The results showed that Drp1 was expressed in layer 5 and 6a of the cerebral cortex, and some expression was also observed in layer 2, although we did not detect obvious Drp1 expression in layer 1 (Fig. 3.A). Drp1 was poorly expressed in the hippocampal region. However, some neurons in the DG-po (polymorphic layer of dentate gyrus) and CA1so (field CA1, stratum oriens) displayed Drp1 expression in the soma and neurite (Fig. 3.C). In the cerebellum, Drp1 protein expression was mainly concentrated in the SIMpu (simple lobule, Purkinje layer). Drp1-positive cells in pu were arranged in a single layer, separating the mo (molecular layer) and the gr (granular layer) (Fig. 3.B). In the spinal cord, high Drp1 expression was observed in the deep layers (VII, VIII, IX, X), which was slightly higher than that in the superficial layers (I, II, III) (Fig. 3.D).
Among all the regions, we selected four regions in which Drp1 was highly expressed (Fig. 4). In the thalamus, Drp1 was highly expressed in the LGd (dorsal part of the lateral geniculate complex) and LGv (ventral part of the lateral geniculate complex) (Fig. 4.A). In the pons, Drp1 expression was higher in the PB (parabrachial nucleus), LC (locus ceruleus) and B (Barrington's nucleus) than in other nuclei (Fig. 4.B). The labeling intensity in the LGd, LGv, PB, CN (cochlear nuclei) and Ⅶ (facial motor nucleus) achieved a strong signal (++++) (Fig. 4.CD). Details of expression of Drp1 protein in all regions are shown in Tab. 1. Moreover, based on the Allen Mouse Brain Atlas [21], we also analyzed the proportion of different Drp1 protein labeling intensities in the brain and spinal cord. The right part of the figure exhibited Drp1 protein expression (Fig. 6), with a low signal (+) accounting for the majority (59% in brain, 60% in spinal cord).
In conclusion, Drp1 protein is widely distributed in the brain and spinal cord, confirming the importance of Drp1 in the CNS.
- At the mRNA level
- Probe titer determination
Using the correctly sequenced recombinant plasmid as the template, the sequence containing the target gene fragment and SP6/T7 promoter joints at both ends was amplified. After amplification, we determined the probe concentration. The Drp1 probe carrying SP6 and T7 was 281.27 ng/µl and 202.61 ng/µl, respectively.
- Identification of probes and antisense probes
Drp1 mRNA gene probes included the upstream and downstream promoter SP6 and T7 sequences, while the sense sequence probes served as the negative control. The results showed a high hybridization signal of SP6 probe, and specific punctate granules were observed, while the T7 probe showed no specific staining. Therefore, the antisense sequence of the target gene Drp1 with SP6 as the promoter was selected for subsequent experiments.
- A wide distribution of Drp1 mRNA in the mouse brain and spinal cord
Results showed that Drp1 mRNA was widely distributed in all mouse brain regions with a strong hybridization signal and high specificity (Fig. 5.A).
In the thalamus, Drp1 was highly expressed in the LGd, LGv and IGL (intergeniculate leaflet of the lateral geniculate complex), with the fluorescence intensity of these three nuclei reaching a high signal (+++) (Fig. 5.H). In the pons, the expression of Drp1 mRNA reached a strong signal (++++) in the PB, LC and B. Expression was lower in the PCG (pontine central gray) than that in these nuclei (Fig. 5.K). In the medulla, Drp1 mRNA was highly expressed in the VCO (ventral cochlear nucleus) and DCO (dorsal cochlear nucleus) (Fig. 5.M). Drp1 mRNA was also widely distributed in the mouse spinal cord, demonstrating a strong hybridization signal (Fig. 5 B). Details of the Drp1 mRNA expression in other regions are shown in Tab. 1 and Fig. 5.
Moreover, we compared the Drp1 labeling intensity at the protein and mRNA levels. Based on the Allen Mouse Brain Atlas[21], the Drp1 signal was visualized in different brain slices. The left part of the figure showed Drp1 mRNA expression, and the right part showed Drp1 protein expression. The results showed that a high intensity Drp1 mRNA labeling signal (+++) accounted for the majority of the expression (53% in brain, 90% in spinal cord), which was higher than the same protein level (+++) in both the brain (10%) and spinal cord (0%) (Fig. 6).
In conclusion, Drp1 was widely distributed in the central nervous system but with heterogeneity, with some areas or nuclei showing high Drp1 expression. Heterogeneity was also found between the Drp1 mRNA and protein level.
- Mainly and highly expressed Drp1 in neurons
The localization of Drp1 in the brain and spinal cord was consistent with what has been proposed in the traditional theory that Drp1 is widely distributed in the CNS[1]. However, unequivocal proof of the innervation is still lacking.
To specify the Drp1 distribution in neurons, we utilized double immunofluorescence to label Drp1 and neurons with the anti-Drp1 antibody and anti-NeuN antibody. Considering all the results, two representative nuclei were selected, and we found that the fluorescence of Drp1 (green) and NeuN (red) coincided well in the VII (Fig. 7.A). However, Drp1 and NeuN were colabeled only in a few parts of the CN, but in other areas of the CN with Drp1 expression, NeuN staining was not found (Fig. 7.B).
In conclusion, Drp1 is mainly and highly expressed in neurons in the central nervous system, and in parts of the nervous system other than neurons, low levels of Drp1 expression could also be observed.
- Highly expression of Drp1 in GABAergic neurons
GABAergic neurons are important inhibitory neurons, which is correlated with the development of many neurological diseases, including Huntington´s disease, Alzheimer´s disease, anxiety, panic disorder and epilepsy[22-24]. To explore the effect of Drp1 on these diseases, we further clarified the distribution of Drp1 in GABAergic neurons (Fig. 1.B). GAD67 (glutamic acid decarboxylase 67)-GFP (green fluorescent protein) transgenic mice were utilized for further observation. In GAD67-GFP transgenic mice, GABAergic neurons are specifically labeled with GFP fluorescence, which will induce spontaneous neuronal fluorescence under a confocal microscope[25-27]. Therefore, colabeling of GABAergic neurons and Drp1 in different brain regions could be observed by GFP and red fluorescence (anti-Drp1 antibody).
We selected three Drp1 and GAD67 double-stained nuclei, including SPVI (spinal nucleus of the trigeminal, interpolar part), ECU (external cuneate nucleus) and SIMpu. The results indicated that Drp1 was overexpressed in the cerebellum Purkinje layer, while Drp1 and GAD67 also colabeled well in this area. Drp1 and GAD67 colabeling was lower in other nuclei (Fig. 8).
- Drp1 was localized not only in mitochondria, but mostly in cytoplasm and dendrites
As we discussed above, the relationship of Drp1 and mitochondria to many neurologic diseases has been confirmed[2]. Moreover, Drp1 is significant in maintaining mitochondrial function through the balanced control of mitochondrial fission and fusion[8]. Therefore, we observed the localization of mitochondrial Drp1 in the brain nucleus. Double labeling of Drp1 (green) and Mito-Red (red) in the LGd, SPIV (spinal vestibular nucleus) and IP (interposed nucleus) showed that Mito-Red completely colocalized with Drp1, while in contrast, the range of the Drp1 distribution was wider than the Mito-Red. Both Drp1 and Mito-Red were scattered around the nucleus. Mito-Red was concentrated on one side of the cell nucleus, while Drp1 expression showed no polarity and connected to form a network structure around the nucleus. In conclusion, the size of the green (Drp1) granules was smaller than the red (Mito-Red) one, but the distribution range of Drp1 was wider than Mito-Red, indicating that Drp1 was localized not only to mitochondria, but mostly in other regions (Fig. 9). Considering that the fluorescent particles may overlap in vertical space, we utilized an electron microscope for further observations (Fig. 1.C)
Next, we investigated the distribution of mitochondrial Drp1 in the PAG (periaqueductal gray) at the subcellular level under an electron microscope. The immune colloidal gold technique was utilized to label Drp1. Colloidal gold was the tracer, which could combine with the positively charged protein molecules in the weak alkaline environment[28, 29]. In Fig. 10, the black particulate matter is colloidal gold particles, which represent positive Drp1 expression (red arrows). The results showed that Drp1 was mainly expressed in the cytoplasmic matrix, which was consistent with our above results. Dendrites could also exhibit some expression, and a small amount of punctate Drp1 expression was distributed on the mitochondrial membrane (Fig. 1.D & Fig. 10.A).
We then further investigated the quantity of mitochondria and Drp1 in axons, axon terminals, dendrites and soma. Seventy axons, 37 axon terminals, 36 dendrites and 6 somas were counted, and the average quantity of mitochondria and Drp1 in each part was calculated. Utilizing the Kruskal-Wallis test, we analyzed Drp1 expression and its correlation with mitochondria from four aspects (Fig. 10.B.C):
1. Quantity of Drp1
The quantity of Drp1 was significantly different in four regions (p<0.001). The quantity of Drp1 was greater in dendrites than axons (p<0.001) and axon terminals (p<0.001). The quantity of Drp1 was greater in soma than axons (p<0.001) and axon terminals (p<0.001)
2. Ratio of Drp1 expression per unit area
The ratio of Drp1 expression per unit area was significantly different in four regions (p<0.001). The ratio was larger in dendrites than axons (p<0.001) and axon terminals (p<0.001).
3. Quantity of mitochondria
The results indicated that the quantity of mitochondria was significantly different in four regions (p<0.001). The quantity of mitochondria was greater in dendrites than axons (p<0.001) and axon terminals (p=0.001). The quantity of mitochondria was greater in soma than axons (p<0.001) and axon terminals (p<0.001).
4. Proportion of Drp1 on mitochondria/total Drp1 in every region and mitochondria with Drp1/total mitochondria in every region
The proportion of Drp1 on mitochondria/total Drp1 in every region was counted. The results showed that in dendrites, 4.72% of the Drp1 was localized to mitochondria (maximum), while in axons this proportion was only 2.5% (minimum)
Similarly, the proportion of mitochondria with Drp1/total mitochondria in every region was also calculated. The results showed that in dendrites, 30.34% mitochondria were localized to Drp1 (maximum), and this proportion was only 14.82% in axonal terminals (minimum). *p<0.05, **p<0.01.
5. Drp1 expression in human malignant glioma tissue reached the highest value in grade III and then declined
As stated above, in components other than neurons, Fig. 7 also showed a low level of Drp1 expression. Glial cells represent another kind of vital component in the brain other than neurons, and some previous research has demonstrated a relationship between Drp1 and glial cells. In models of ischemic injury, Zhou et al demonstrated an upregulation of Drp1 and enhanced mitochondrial fission in microglia[30]. Chae et al revealed that Drp1-mediated mitochondrial fission could activate microglial cells by regulating p62-mediated autophagy[31]. Considering the crucial role of Drp1 in glial cells, we speculated that the other regions expressing Drp1 were probably glial cells.
Therefore, we also analyzed Drp1 expression in human malignant glioma tissue. With the normal human brain as the control tissue, grade I represented juvenile glioma tissue and grade II-IV represented glioma tissue with mild, moderate and severe malignancy, respectively.
Eight fluorescent spots were randomly selected in every tissue grade. The Drp1 fluorescence intensity was calculated using ImageJ software (Tab. 2&Fig. 11). One-way ANOVA was used for the statistical analysis. The results revealed significantly different Drp1 expression in glioma grade I-IV (p=0.01). After the LSD (least significant difference) test, Drp1 expression was higher in the control group than grade I (p=0.018). In contrast, it was higher in grade III than the control group (p=0.036), grade I (p<0.001), grade II (p=0.011), and grade IV (p=0.001). * p<0.05, ** p<0.01.
In conclusion, from grade I-III, the mean Drp1 fluorescence intensity showed an increasing trend. The mean Drp1 fluorescence intensity was significantly reduced in grade IV (Fig. 11.C)