Autophagy Review—Li Xiaojiang's team reviews the differences and research progress of mitochondrial autophagy in vivo and in vitro models

Parkinson disease (PD) is the second largest neurodegenerative disease after Alzheimer's, affecting about 1-2 people per 1,000 people
.
The prevalence of Parkinson's disease increases with age, with a prevalence of about 1% in people over 60 years
of age.
Its main neuropathological change is the formation of many Lewy bodies containing α-synuclein (α-synuclein) in the brain area of the substantia nigra of the midbrain.
In addition, dopaminergic neurons in the substantia nigra are greatly lost, which in turn presents with dyskinesia, including three major symptoms: tremor, rigidity, and bradykinesia [1] (recently revised diagnostic criteria exclude postural instability as a fourth marker).

The pathogenic factors of PD are complex, and genetic, environmental, ethnic, and even gender factors will affect
the pathogenesis of PD.
According to the cause of the disease, PD can be divided into two categories: sporadic and hereditary, of which 5-10% of patients are caused by genetic mutations
.
Mutations in many genes have been reported to cause PD, such as PINK1, PRKN, DJ1, LRRK2 , SNCA, VPS35, GBA, etc
.
However, the pathogenesis of PD is still unclear
.

Mitochondria, as the energy factory of cells, participate in many important physiological processes
such as cell metabolism, signaling, differentiation, growth, apoptosis and death.
When mitochondria are damaged, pro-apoptotic factors can be released to cause cell death
.
Mitophagy is the mechanism in which cells selectively remove damaged mitochondria through the mechanism of autophagy to protect cells
.
PD has long been thought to be associated with mitochondrial damage [2], and PINK1, as a mitochondrial membrane protein, is able to sense mitochondrial damage and is recruited to activate downstream E3 on damaged mitochondria The ubiquitin ligase PRKN and ubiquitin, forming a complex that enables damaged mitochondria to be cleared by lysosomes in time, thereby protecting nerve cells
.
This classical theory is mainly based on a large number of in vitro experiments
.
However, PINK1 is difficult to detect in mouse brains, and changes in wireless autophagy function in mouse with PINK1 knockout cannot mimic the pathological features of nerve cell death in patients' brains [3-6].

Therefore, how PINK1-PRKN regulates mitophagy and its association with PD under physiological and pathological conditions in vivo remains unclear
.
In recent years, new evidence has shown that the loss of PINK1 in the brains of nonhuman primates causes severe nerve cell death but does not affect mitochondria [7,8].

However, there is still a lack of systematic research reviews to evaluate
the large differences between PINK1-mediated mitophagy in vitro experiments and in vivo experiments in animal models and their potential mechanisms.
Therefore, it is of great significance to deeply explore and summarize the differences between PINK1-PRKN-regulated mitophagy in in vivo studies in animal models and in vitro studies in cell models.

On October 26, 2022, Professor Li Xiaojiang's team from the Guangdong-Hong Kong-Macao Institute of Central Nervous Regeneration of Jinan University was invited to the internationally renowned journal Autophagy.
A review entitled "PINK1-PRKN mediated mitophagy: differences between vitro and in vivo model
" was published online.
Han Rui, a doctoral student in Li Xiaojiang's team at the Guangdong-Hong Kong-Macao Institute of Central Nervous Regeneration of Jinan University, is the first author of the paper, and researcher Yang Weili is the corresponding author
of this review.
The authors focus on comparing the main differences between PINK1-PRKN-mediated mitophagy in vitro and in vivo models, and discuss the reasons for these differences, aiming to understand how PINK1 performs its function in different environments, and thus to study PINK1 The important function of PRKN in the primate brain provides new research ideas
for exploring the mechanism of PD pathogenesis caused by mutations between the two and finding possible treatment strategies.

Acute exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine , MPTP) causes Parkinson's symptoms as well as the death of dopamine neurons [9], and its metabolite MPP+ acts as a mitochondrial complex I Inhibitors are selectively absorbed by dopaminergic neurons and cause mitochondrial dysfunction [10-12].

This result is the first to confirm the relationship
between selective neurodegeneration of PD and mitochondrial dysfunction.
In addition to MPTP, other mitochondrial poisons, such as rotenone and paraquat, are associated with an increased risk of PD [13-15].

In the brains of PD patients who have died, there are many changes associated with mitochondrial dysfunction [16,17].

The above conclusions support the thesis that dopaminergic neurons are more sensitive to mitochondrial dysfunction and that there is a strong correlation between PD and mitochondrial dysfunction.

PINK1 and PRKN are the pathogenic genes of two early-onset Parkinson's diseases, with the characteristics of common-stained recessive inheritance, and the loss of protein function caused by the mutation of the two will cause PD, but the pathogenic mechanism is not clear, and some studies believe that it is related to mitochondrial dysfunction [18-20]
。 PINK1 is a mitochondrial serine, threonine kinase, and PRKN is an E3 ubiquitin ligase
.
In genetic experiments on fruit flies, PRKN was found to be located downstream of PINK1 [21-23].

Later, biochemical analysis found that the two have the function of
sensing mitochondrial damage and recruiting ubiquitin system-related proteins to remove damaged mitochondria together.

The mitochondria-stimulating drug carbonyl cyanide m-chlorophenyl hydrazine (CCCP), antimycin A, were added to many cell lines and mouse embryonic fibroblasts (antimycin A) or rotenone, it can cause the accumulation of PINK1 on the outer mitochondrial membrane, and then further recruit PRKN and activate its E3 ubiquitin ligase activity, The proteins of the damaged mitochondria are then ubiquitinated together with ubiquitin and further transferred to lysosomes for removal [19,24-28].

Together, these conclusions suggest that PINK1 has the effect of protecting mitochondrial dysfunctional cells from apoptosis caused by environmental stimuli (Figure 1).

However, PINK1-PRKN-mediated mitophagy was found to be a rather slow process in cultured mouse cortical neurons, indicating that PINK1 and PRKN regulate mitochondrial autophagy with cell type dependence
.
This may be due to the fact that the production mode of immortal cells is glycolysis and the production mode of nerve cells is oxidative phosphorylation, and the two have different degrees of dependence on mitochondria, resulting in different ways of causing mitochondrial autophagy, and there may be other pathways in nerve cells that regulate mitochondrial homeostasis
.

Fig.
1 In vitro study of PINK1-PRKN-mediated mitochondrial autophagy

(Source: Han R, et al.
, Autophagy, 2022).

In vitro experiments have many limitations - including exogenous overexpression of PINK1, PRKN, or acute mitochondrial injury by administration, and the results of these methods are inconsistent with the expression levels of endogenous proteins under normal conditions in vivo, and are also related to physiological, The degree of stress on mitochondria in pathological conditions is inconsistent
.
Therefore, there is an urgent need for suitable animal models
for the study of PINK1-PRKN-mediated mitophagy.
In fact, as early as 2003, scientists confirmed that PINK1-PRKN can synergistically regulate mitochondrial quality in fruit flies, and PRKN-deficient flies show phenotypic and pathological changes of dyskinesia, muscle degeneration, and mitochondrial dysfunction [21]
。 Subsequent studies of Drosophila confirmed that PRKN was on the same pathway as PINK1 and that PRKN was located downstream
of PINK1.
A series of experiments in Drosophila demonstrated the ability of PINK1-PRKN to work together to maintain mitochondrial integrity in energy-intensive tissues [22,23].

Although PINK-PRKN-mediated mitophagy can be detected in vitro experiments as well as in fruit fly experiments, whether this role exists in the mammalian brain is an important question
to be answered in the field.
As a result, scientists have spent a lot of effort to build many rodent models of PINK1 and PRKN knockouts, but none of the mouse models have shown PD-like dyskinesia and neuronal degeneration [3,4]
。 Loss of age-dependent mild TH-positive neurons has been reported in the pink1KO rat model, but has not been consistently concluded in other studies [29,30].

Experiments with the help of mito-QC and mt-Keima reporting system found that pink1 and prkn deficient mice did not show obvious defects in mitochondrial autophagy [5,6,31]
。

Because mouse models cannot mimic the pathological changes in neuronal loss in PD, and there are no significant changes in mitochondrial autophagy, the researchers considered using more advanced large animals to build PD models
.
In recent years, scientists have used CRISPR/Cas9 technology to establish the PINK1/PRKN knockout pig model, but the pig model has not shown significant neurodegeneration and dyskinesia [32,33].

Therefore, the pathological changes caused by the deletion of PINK1-PRKN may have a strong species dependence, which forces scientists to use non-human primates to establish monkey models of PINK1 and PRKN deletions [35].

。 After unremitting efforts, the authors' team used CRISPR/Cas9 technology to establish a PD monkey model of PINK1 knockout, and found that PINK1 was knocked out at the embryonic stage The characteristics of severe nerve cell death in newborn target monkeys [34], so the severe neuronal loss in the brain of the PINK1 mutant monkey contrasts sharply with the absence of neurodegeneration in a mouse model that completely knocked out the PINK1 gene
。 Importantly, PINK1 is only expressed in primate brains, and the loss of PINK1 does not cause changes in mitochondrial morphology and metabolic function in monkey brains, but reduces the phosphorylation levels of a series of neurologically related proteins and causes nerve cell death [8] 2）
。 Therefore, the establishment of the non-human primate monkey model is of great significance
for studying the function of PINK1 in the primate brain and the neuropathological mechanism of PD induced by its loss.

Figure 2 In vivo study of PINK1

(Source: Han R, et al.
, Autophagy, 2022).

In vitro models cause disruption of mitochondrial membrane potential by adding mitochondrial poisons, but is there such acute, excessive stimulation in the in vivo situation? Neurodegeneration with age during PD may be associated with
chronic mitochondrial damage.
But whether this chronicly accumulated stimulation causes PINK1-PRKN-mediated mitophagy remains unclear
.
In addition to this, mitochondrial metabolic function and the situation of mitochondrial autophagy are not the same
in different types of cells.
For example, nerve cells rely heavily on oxidative phosphorylation for energy, which has a lethal effect
on cells when mitochondria are lost in large quantities.
Immortal cells generally come from cancer cells, which rely on glycolysis for energy, so when a large number of mitochondria are lost, they are better
tolerated.
Thus, in experiments with the addition of CCCP drugs to induce mitochondrial damage in immortal cell lines, PRKN was able to shift onto mitochondria, which is difficult to detect in cultured primary neurons [36,37].

More importantly, endogenous PINK1 could not be detected in cell lines and mice, but had specific and abundant expression in non-human primate monkey brains, and PINK1 was found to play an important kinase role as a cytoplasmic protein in primate brains [ 8]
。

The differences in PINK1-PRKN function in vivo and in vitro models raise a number of important scientific questions that researchers should further address
.
First, why is PINK1 undetectable in mice and abundantly expressed in monkey brains? PINK1mRNA is widely and richly expressed in different species, so perhaps protein translation or/or protein stability levels have different regulatory patterns in different species, which needs to be studied
by rigorous experiments.
Secondly, to what extent does PINK1-PRKN-mediated mitophagy function in vivo in pathological states? In primate brains, when chemicals induce mitochondrial damage, whether full-length PINK1 accumulates on mitochondria, and how cytoplasmic PINK1 works, these questions need to be answered
in more depth.

In this review, the authors focus on the main differences between PINK1-PRKN-mediated mitophagy in vitro and in vivo models, and discuss the reasons for these differences, with the aim of understanding how PINK1 performs its function in different environments, and thus for the study of PINK1 The important function of PRKN in the primate brain, exploring the mechanism of PD pathogenesis caused by two mutations and finding possible treatment strategies provide new research ideas
.
At the same time, based on the characteristics of PINK1 expressed only in primate brains, the authors point out that non-human primate monkeys are irreplaceable and important animal models
to study the in vivo function and mechanism of action of PINK1-PRKN.

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Dr.
Han Rui (left), researcher Willy Yang (right).

(Photo provided by: Li Xiaojiang's research group, Guangdong-Hong Kong-Macao Institute of Central Nervous Regeneration, Jinan University)