1. Drp1 widely but heterogeneously distributes in central nervous system.
This study reveals that Drp1 is widely distributed in central nervous system at both protein and mRNA level, which firmly demonstrates the importance of Drp1. Moreover, Drp1 is widely expressed in the cytoplasm but scarely distributed on mitochondria, which only accounts for about 5%. This result is consistent with previous studies[19, 20]. However, Drp1 distribution also shows the heterogeneity.
1.1 The expression of Drp1 in different kinds of neurons is heterogeneous.
In inhibitory neurons, we found high Drp1 expression. Figure 4 showed that Drp1 was highly expressed in the dendrites of the Mo layer (the dendrites of the Purkinje cell layer) in the cerebellar cortex. The neuron in this layer is the important inhibitory neuron[21]. In addition, Drp1 is also expressed in the second layer of the cerebral cortex, which is the external granular layer[22]. Drp1 expression difference in cerebral cortex may be related to the diverse cell types of each layer. The second layer contains a large number of granular cells, the majority of which are GABAergic inhibiting neurons[23].
1.2 Drp1 expression in neurons also shows heterogeneity.
All immunofluorescence results showed that Drp1 was highly expressed and distributed around the nucleus, and IEM results showed that Drp1 was mainly expressed in dendrites. In addition, we found that Drp1-positive protuberant structures (long strips in Fig. 4), may possibly be the axons or dendrites. Therefore, it can be speculated that the distribution of Drp1 is mainly concentrated in the dendrites.
As the vital factor in regulating mitochondrial fission process, Drp1 should be expressed in all cells. However, as is shown in Fig. 9, Drp1 protein and mRNA expression is different in same region, and no Drp1 expression was found in some neurons, which might suggests that some other molecules could also induce mitochondrial fission process. Further investigation is still needed.
2. Drp1 heterogeneous distribution may contribute to the occurrence and development of neurological diseases.
As we discussed above, mitochondrial dysfunction is involved in the occurrence and development of neurologic disease[24]. Drp1 mutation promotes mitochondrial dysfunction[25]. A large number of researches also demonstrated that GABA is related with the development of neurological diseases.
GABA is the major inhibitory neurotransmitter in CNS. It is confirmed that dysfunction of GABA metabolism or GABAergic neurons is associated with many neurological diseases, including Huntington´s disease, Alzheimer´s disease, anxiety, panic disorder and epilepsy[26, 27]. However, whether Drp1 directly affects these diseases through GABAergic neuron is still unclear.
Therefore, we analyzed the Drp1 expression in GABAergic neurons. The results of this study showed that Drp1 distribution is mainly concentrated in the initial part of GABAergic neurons, dendrites, axons and neuron mitochondria. This heterogeneous Drp1 distribution may contribute to neurological diseases occurrence and development.
As we discussed in introduction, some molecules targeted to Drp1 have been found. However, the impact of the molecules mentioned above on human body and in what diseases these molecules be can used in clinic still need to be further studied. Therefore, heterogeneous Drp1 distribution in GABAergic neurons may give us the targeted treating guidance toward the GABA related diseases.
3. Drp1 expression in human malignant glioma further demonstrates Drp1’s significance in cells.
Based on the results above, we make the conclusion that Drp1 widely but heterogeneously distributes in central nervous system, which firmly indicates the importance of Drp1. However, in central nervous system, the connection between glial cells and neurons also plays the vital role in regulating normal brain function. In Fig. 10, alongside neurons, we found that in other parts of the nervous system such as glial cells, small amount of Drp1 expression could also be observed.
Therefore, we further analyzed Drp1 expression in human malignant glioma tissue. Compared the mean value of Drp1 fluorescence intensity in every grade, we found the increasing Drp1 fluorescence intensity trend from grade I-III. In grade IV, mean value of Drp1 fluorescence intensity significantly reduced. The highest fluorescence intensity in moderate malignancy indicated that the mitochondrial dynamic changes reach the maximum. In grade IV, normal tissue was destroyed, so fluorescence intensity significantly reduced. Such expression changes were also found in other malignant tumors[28, 29]. Thus, results of Drp1 expression in human malignant glioma further demonstrate Drp1’s significance in cells, and may give us the guidance that Drp1 changes could function as the early indicator of malignant diseases.
4. Inhibiting Drp1 may be the novel target for neurological diseases treatment.
When mitochondrion is going to divide, Drp1 will translocate from cytoplasm to mitochondrial outer membrane with the help of receptors and adapters[30], and assemble into ring-like structures on mitochondrial outer membrane, leading into mitochondrial fission[31, 32]. When Drp1 translocates abnormally to the mitochondrial membrane, mitochondrial dynamic homeostasis regulated by Drp1 will be broken, leading to mitochondrial morphological changes[17]. Therefore, inhibiting the Drp1 harboring in neurons may be the novel target in treating the neurological diseases[33].
For treatment, we identify three molecules which can inhibit Drp1. P110 can effectively inhibit the translocation of Drp1 from cytoplasm to mitochondria, and inhibit the binding of Drp1 to Fis1, thus inhibiting mitochondrial division[17]. Hirotsugu Kanda et al found the significant increase of Drp1 in neuropathic pain model. Further study demonstrated that intrathecal Drp1 antisense oligodeoxynucleotide (ODN) could decrease the spinal Drp1 expression[34]. Besides ODN, mdivi-1 is another Drp1 inhibitor. Luiz F also revealed that mdivi-1 could attenuate the neuron pathologic changes[16].
Nowadays, there is still no better method for the prevention of neurological diseases like the AD, HD. And the disease diagnosis often lags behind the disease development. Therefore, new target drugs need to be developed urgently. It is clear that P110, mdivi-1[35, 36] and OND can effectively inhibit Drp1 translocation from cytoplasm to mitochondria. However, the impact of the molecules mentioned above on human body and whether it can be efficiently and conveniently applied in clinic still need to be further studied.
This study also needs improvements in several parts. It is much better to use Drp1 knockout mice in the investigation of Drp1 distribution, but these Drp1 knockout mice can't survive. Moreover, the phosphorylation status of Drp1 is essential for its interaction with Mff, and takes the vital role in regulating mitochondrial dynamic, but this study didn’t observe the phosphorylation Drp1 status. We will investigate it in the following research.
In this study, we observed the Drp1 distribution in brain and found that Drp1 widely yet heterogeneously distributes in central nervous system. Drp1 heterogeneous distribution may be involved in the occurrence and development of neurological diseases. Moreover, we identified three targeted molecules for treatment. We hope that this research on the relationship between Drp1 and mitochondria in neurons may be helpful in the molecular therapy, and give the clinical guidance for neurological diseases treatment. As the vital factor for mitochondrial dynamics, future studies are still needed on the Drp1 distribution and translocation beyond the pathological changes.