New target for mitochondrial protection: Butylphthalide improves cognitive function in white matter diseases by reducing demyelination

Recently, the latest research result "Dl-3-n-Butylphthalide Alleviates Demyelination and Improves Cognitive Function by PromotingMitochondrial Dynamics in White Matter Lesions" by Professor Dong Qiang's team from Huashan Hospital affiliated to Fudan University was published online in Frontiers in Aging Neuroscience (IF: 4.
362).

The study explored the effects of butylphthalide (hereinafter referred to as "NBP") on the cognitive function and nerve demyelination of white matter lesion model mice (hereinafter referred to as "WML"), and proved that NBP can pass WML as a potential therapeutic target ——Inhibit mitochondrial toxoplasmin (SNPH) to play a neuroprotective effect, and at the same time, by promoting changes in mitochondrial dynamics, significantly alleviate the degree of demyelination of the WML model and improve its spatial learning ability and memory function.

Research background White matter lesions (WMLs) are cerebrovascular diseases accompanied by demyelination and cognitive decline.

Especially in the elderly, this pathological change is one of the main factors leading to cognitive decline and vascular dementia (VaD).

Ischemia and hypoxia caused by long-term brain tissue hypoperfusion is an important reason for its production, and nerve demyelination is also an important pathological feature that leads to impaired cognitive function and poor clinical prognosis in patients.

Therefore, we need to urgently find a way to reduce the changes in demyelination and protect the function of the cranial nerve.

In the case of cerebral hypoperfusion, mitochondria, as the most sensitive organelle in neurons, can sense ischemia or hypoxia and rapidly change its dynamics and metabolism.

When axons are damaged by ischemia, the mitochondria will have abnormal dynamics, the division, fusion, and transport functions of axons will be disordered, and the anti-transport function of cells will also be reduced.
This will block normal mitosis of cells and produce harmful effects.
Reactive oxygen species (ROS) damage the myelin sheath.

The anterograde of mitochondria is essential for providing energy to synapses and releasing neurotransmitters.

With reference to previous research results, the study speculated that NBP may reverse the harm of WML due to demyelination by regulating mitochondrial dynamics.
Therefore, in this study, the WML model was used to study the effect of NBP on mitochondrial dynamics and demyelination.

Research methods C57BL/6J male mice (9-12 weeks, 25-30 g) were modeled with bilateral carotid artery stenosis (BCAS).

Divided into sham operation group and butylphthalide treatment group.

In the butylphthalide group, it is divided into a low-dose treatment group (L-NBP, 50 mg/kg/day) and a high-dose treatment group (H-NBP, 100 mg/kg/day), which will be performed 1 day after BCAS Gavage for 28 days.

All experimental groups were randomly divided into groups, and all results analysis were conducted by independent researchers who did not understand the treatment conditions and mouse models.

Research results 1.
NBP reduces the demyelination of the BCAS model and improves cognitive function.
Transmission electron microscopy (TEM) is used to evaluate the myelination status of different treatment groups.
The whole brain hypoperfusion of BCAS changes the total axon G-ratio distribution.
It has a higher G-ratio distribution rate, and at the same time, the G-ratio of the BCAS group increases significantly.

Although low-dose NBP treatment (L-NBP, 50 mg/kg/day) did not restore demyelination, high-dose NBP treatment (H-NBP, 100 mg/kg/day) alleviated the demyelination caused by BCAS Sheath (Figure 1A–C).

Through the eight-arm radial maze test, the therapeutic effect of NBP on BCAS-induced cognitive impairment was further evaluated.
In the BCAS model group, mice showed higher revisit errors and lower revisit errors in the first eight-arm path selection.
Different arm choices indicate that the working memory of BCAS mice is significantly impaired, and the high-dose NBP treatment group can significantly reduce the working memory impairment caused by BCAS modeling (Figure 1D-F).

2.
NBP reduces white matter demyelination and cognitive impairment by reducing the accumulation of mitochondria between axons.
TEM was used to detect the mitochondrial load between axons.
It was found that the mitochondrial load in axons in the BCAS group was significantly increased, accompanied by abnormal mitochondrial morphology.

High-dose NBP treatment can significantly reduce the mitochondrial load of BCAS mice and reduce the abnormal morphological changes of mitochondria (Figure 2A-D).

Although motor proteins, such as Miro and Milton, did not show significant changes after BCAS, SNPH, which anchors mitochondria to microtubules, showed a significant increase after BCAS, which was significantly reduced by high-dose NBP treatment.
The expression of SNPH indicates that high-dose NBP may reduce mitochondrial accumulation by inhibiting the expression of SNPH (Figure 2E, F).

3.
NBP reduces mitochondrial damage by promoting mitochondrial dynamics.
In order to further clarify the mechanism of NBP to reduce mitochondria, an in vitro hypoperfusion model was established in a low-sugar and low-oxygen environment.

Studies have found that low glucose and hypoxia treatment will significantly increase the mitochondrial load, accompanied by a decrease in mitochondrial dynamics, while NBP treatment can rescue the decline in mitochondrial dynamics caused by low glucose and hypoxia, and reduce the load of mitochondria in axons.

The high-dose NBP group reduced the mitochondrial load by inhibiting the expression of active SNPH in the body.
In addition, on the culture slices overexpressing SNPH, it was found that the mitochondrial load reduced by the high-dose NBP was offset by SNPH OE (Figure 3A-D).

4.
NBP enhances the level of mitochondrial dynamics, reduces the production of ROS in axons and reduces demyelination.
Studies have found that the production of ROS increases after low glucose and hypoxia treatment, but NBP can significantly reduce the production of ROS and total ROS in axons (Figure 4A) -C). The expression of MBP and NF and the ratio of MBP/NF were significantly reduced in the hypoglycemia and hypoxia group, which impaired myelination.

NBP inhibits the production of demyelination through the SNPH pathway.

NBP treatment reduced the production of ROS between axons and rescued nerve demyelination, which is related to the improvement of mitochondrial reverse transport.

 5.
NBP restores the synaptic signals of damaged neurons and enhances mitochondrial dynamics.
NBP treatment restores the intrinsic excitability of neurons, while SNPH OE eliminates the therapeutic effect of NBP to a certain extent (Figure 5A–C).

After further testing the changes of synaptic signal after low glucose and hypoxia treatment, it was found that the amplitude and frequency of mEPSC were significantly reduced, and high doses of NBP could reverse this phenomenon, but SNPH OE would offset these therapeutic effects (Figure 5D-F).

The cumulative mEPSC distribution curve analysis showed that the NBP treatment established a significantly richer mEPSC amplitude and a smaller mEPSC interval (Figure 5G, H).

These results indicate that NBP promotes intrinsic excitability and synaptic function of neurons, which is related to mitochondrial forward transport.

6.
NBP reduces demyelination and cognitive impairment by inhibiting SNPH.
The increase in G-ratio after SNPH OE in the high-dose NBP treatment group shows that SNPH OE significantly eliminated the therapeutic effect of NBP to reduce demyelination (Figure 6A, B).

At the same time, SNPH OE also inhibited the reduction of mitochondrial load and the restoration of mitochondrial morphology in the high-dose NBP treatment group (Figure 6C–E).

In addition, the synapses increased by high-dose NBP treatment were lost in the NBP+SNPH OE group, accompanied by a decrease in mushroom-shaped synapses (Figure 6F–H).

The memory impairment improved by NBP was impaired by SNPH OE (Figure 6I–K).

The conclusions of the study indicate that the increased expression of axon-specific toxoplasmin SNPH will inhibit mitochondrial dynamics, and axon ischemia and hypoxia damage will lead to static mitochondrial accumulation.

NBP treatment improves the forward and reverse transport of mitochondria and reduces the accumulation of mitochondria in axons, thereby preventing the destruction of myelin and improving the electrical conduction function of synapses.

In addition, NBP treatment also increased the number of nerve synapses and improved cognitive function, revealing that NBP can inhibit the expression of SNPH, and SNPH OE can counteract the protective effect of NBP in reducing demyelination and improving cognitive function.

It can be seen that NBP is a promising treatment method to alleviate the cognitive dysfunction and demyelination of WML.

 References: YiweiFeng, Min Guo, Qiang Dong, et al.
Dl-3-n-Butylphthalide Alleviates Demyelinationand Improves Cognitive Function by Promoting Mitochondrial Dynamics in White MatterLesions.
[J].
Front.
Aging Neurosci.
, 08 March 2021.