Nature & Cell article interpretation: Relationship between ion channels on the surface of lysosomal membranes and Parkinson's syndrome

As a "recycling station" in the cell, lysosomal plays a vital role in maintaining cell homeostasis, Xiaoyou once introduced the relationship between lysosomal degradation (autophagy) and neurodegenerative diseases, interested friends please poke here to understand

Figure 1.

Figure 2.

Fig.

Figure 4.

Figure 5.

Figure 6.
A conformational change of AKT can activate the TMEM175 channel (GF= growth factors)

Next, the authors studied two common minor alleles of TMEM175, rs34311866 and rs34884217, corresponding to the M393T and Q65P mutations
of the TMEM175 channel, respectively.
According to studies, the mutation of the former was positively correlated with the onset and progression of PD, while the latter was negatively correlated
.

The electrophysiological results of Figure 7(d) show that (M393T mutation) has a significant reduction in current in the mutant model compared to the wild-type model (43% lower in basal cells and 54% lower in sc79 activated cells).

For the Q65P mutation, the current disappeared after 3 hours of starvation in the wild-type samples, and the mutant samples disappeared completely after 6 hours (Figure 6), indicating that the mutated model was significantly more
tolerant to extracellular growth factors.

These results suggest that the two mutations—M393T and Q65P—are of different types, with the former being a loss of function type of lysosomal potassium channels and the latter being a function-acquiring mutation
.
At the same time, the supplementary data of the article also show that compared with the wild type, the neurons after TMEM gene knockout are significantly less resistant to harsh conditions such as neurotoxins (MPTP), while the mutation model of Q65P can resist this damage, further illustrating that such lysosomal potassium channels have a protective effect
on nerves.

Fig.
7.
Bidirectional regulation of
lysosomal potassium ion channels by common TMEM175 mutations (M393T and Q65P) associated with predisposition to Parkinson's disease.

So how is this lysosomal potassium channel in the study related to Parkinson's disease? Alpha-Synuclein (α-synuclein, α-syn) accumulation is one
of the hallmarks of PD.
As shown in Figure 8, through immunofluorescence and histochemistry experiments, the authors observed significant α-syn accumulation in mice after TMEM175 knockout, and the partially reduced sample of TMEM175 (about a 50% decrease) also accelerated the aggregation
of this protein.
These results suggest that the deletion of TMEM175 impairs dopaminergic neurons and their motility in
mice.

Figure 8.
Lack of potassium channels in lysosomals can lead to accelerated spread of pathogenic α-syn in mice, loss of dopaminergic neurons, and impaired
motor function.

The paper also conducted clinical validation (Figure 9), which showed that Parkinson's patients with the M393T mutation had significantly lower cognitive and motor abilities (compared to patients without this mutation
).

Figure 9.
Results of clinical studies of M393T mutations on the course of Parkinson's disease

PART TWO: TMEM175 associated with Parkinson's disease belongs to the lysosomal hydrogen ion channelIn June this year, the team of the article found that under physiological acidic conditions, the ions through TMEM175 were mainly hydrogen ions, not potassium ions, and through a series of quantitative analyses, it was found that TMEM175 was about 50,000 times more permeable to hydrogen ions under this condition than potassium ions
。

The author's inspiration for this study was a finding that when directly electrophysiological recording of intracellular lysosomal membranes, as long as there is a difference in pH between the inside and outside of the lysosomal membrane (neutral environment with pH = 7.
2 outside the membrane, and pH = 4.
6 acidic environment inside the membrane) can always be recorded from the membrane to the outside of the membrane
, as long as the physiological conditions are imitated.
If the in-membrane pH is adjusted to a more acidic 3.
5, this signal will be stronger
.

Based on this finding, the authors made the following bold assumptions: the ion channel that mediates this current is the hydrogen ion channel on the lysosomal membrane that the scientific community is looking for, and cooperates with the V-ATPase proton pump to adjust the pH balance
of the lysosome one in and one out.

Therefore, to identify this desired hydrogen channel, the authors performed a series of possible lysosomal membrane proteins (in addition to TMEM175, TPC1/TRPML1/CLCN4/CLN8, etc.
) one by one overexpression tests (Figure 10).

It was found that only when TMEM175 was overexpressed, the recorded current was about
20 times higher than other membrane proteins.
At the same time, the sample after being knocked out by the gene has no observable current
even if the pH is set to a lower value (3.
5).
These conclusions indicate that TMEM175 is the lysosomal hydrogen ion channel
to be sought.

Figure 10.
TMEM175 is necessary and sufficient for proton permeation currents activated by lysozyme plasmodium.

At this point in the study, the conclusion that contradicted previous studies was found, that is, previous studies generally believed that TMEM175 was a potassium ion channel, and then the article further studied
the differences between the two.
After careful comparison, it was found that the previous literature did not consider the regulatory effect of pH on the function of TMEM175, so it will be assumed that TMEM175 is mainly permeable to potassium ions
.

Therefore, the authors conducted a series of studies that set the internal environment of the lysosomal in a pH environment close to the physiological conditions (Figure 11), and found that TMEM175 has stronger hydrogen ion permeability (about 50,000 times that of potassium ions)
under this pH condition.

 

Figure 11.
The TMEM175 channel has strong proton passability

In addition to this, the study also identified that the small molecule compounds DCIB and ML 67-33 can also activate the TMEM175 channel
.
These compounds can activate not only the hydrogen ion current in the channel under acidic conditions, but also the potassium ion current under neutral conditions (Figure 11
).
In addition, the study also found that the lipid molecule Arachidonic Acid (ArA) is an endogenous activator of the TMEM175 channel, indicating that the lipid signaling pathway is also likely to have a regulatory effect
on the activity of TMEM175.

Figure 12.
Arachidonic acid (ArA) and synthetic chemicals can activate TMEM175

At the cellular level, the study found that the pH homeostasis of the intracellular lysosome after knockout of the TMEM175 gene was disrupted, and the lysosome was in an overly acidic state at this time, and the activity of the proteolytic enzymes Cathepsin B and Cathepsin D inside the lysosome was also affected by immune cell experiments (Figure 13
).

 

Figure 13.
Proton permeation through TMEM175 is required for lysosomal pH equilibrium and efficient proteolytic degradation

Then the researchers derived from the cellular level to the mouse organism, constructed an animal model of TMEM175 gene knockout, as shown in Figure 14, in the mouse neuronal tissue also observed the phenomenon
of lysosomal acidity is too strong, lysosomal degradation function impaired.
At the same time, the accumulation of α-Synuclein (mentioned earlier) associated with the pathogenesis of the neurodegenerative disease Parkinson's disease is also more pronounced in TMEM175 knockout mice, which is speculated to have a further impact on
the condition of PD.

 

Figure 14.
Deficiency of mouse neuronal TMEM175 leads to excessive acidification of lysosomals, impaired hydrolytic activity of lysosomals, and accumulation of α-Synuclein nuclear proteins in mouse brains

Both articles provide a broader idea for the study of PD, especially the second, revealing that TMEM175 is a novel hydrogen ion-activated hydrogen ion channel
.
In recent years, researchers have widely detected mutations in the TMEM175 gene in patients with Parkinson's syndrome, and in the future, Xiaoyou predicts that there will be more wonderful studies around the regulatory mechanism of TMEM175 and how TMEM175 affects the neurodegenerative model has been published
.

At the same time, many research teams are also developing targeted drugs for regulating lysosomal ion channels, including TRPML1 and TMEM175, which may be used to prevent and treat neurodegenerative diseases related to metabolic degradation of lysosomes in the future, such as Parkinson's disease and Alzheimer's disease
.