Wild-Type DCTN1 Suppresses the Aggregation of DCTN1 Mutants Associated with Perry Disease

Abstract

Perry disease, a rare autosomal dominant neurodegenerative disorder, is characterized by parkinsonism, depression or apathy, unexpected weight loss, and central hypoventilation. Genetic analyses have revealed a strong association between point mutations in the dynactin I gene (DCTN1) coding p150^glued and Perry disease. Although previous reports have suggested a critical role of p150^glued aggregation in Perry disease pathology, whether and how p150^glued mutations affect protein aggregation is not fully understood. In this study, we comprehensively investigated the intracellular distribution of the p150^glued mutants in HEK293T cells. We further assessed the effect of co-overexpression of the wild-type p150^glued protein with mutants on the formation of mutant aggregates. Notably, overexpression of p150^glued mutants identified in healthy controls, which is also associated with amyotrophic lateral sclerosis, showed a thread-like cytoplasmic distribution, similar to the wild-type p150^glued. In contrast, p150^glued mutants in Perry disease and motor neuron disease caused aggregation. In addition, the co-overexpression of the wild-type protein with p150^glued mutants in Perry disease suppressed aggregate formation. In contrast, the p150^glued aggregation of motor neuron disease mutants was less affected by the wild-type p150^glued. Further investigation of the mechanism of aggregate formation, contents of the aggregates, and biological mechanisms of Perry disease could help develop novel therapeutics.

INTRODUCTION

Perry disease, a rare autosomal dominant neurodegenerative disorder, is characterized by Parkinsonism, depression or apathy, unexpected weight loss, and central hypoventilation.¹⁾ The mean age of disease onset is 49 years, and the number of patients worldwide is <100.^2,3) The average disease duration from onset to death is 5 years,^2,3) indicating a poor prognosis for this disease. The most common initial symptoms are depression or apathy, and the most common causes of death are respiratory failure or pneumonia caused by hypoventilation.³⁾ In addition, nocturnal hypoventilation is observed in the late stages of the disease, leading to respiratory insufficiency, insomnia, and ultimately death within 2–10 years.⁴⁾ Genetic analyses have revealed a strong association of missense mutations in the dynactin I gene (DCTN1) with Perry disease, including F52L, G67D, G71A, G71E, G71R, T72P, Q74P, and Y78C, located in exon 2 of DCTN1.^3,5–12) Disease penetrance has not been precisely determined because of the limited number of patients; however, all asymptomatic heterozygotes were younger or within the range of onset age at the time of diagnosis,¹³⁾ indicating a high penetrance of DCTN1 mutations in Perry disease. DCTN1 encodes the dynactin subunit p150^glued, a vital factor in the dynactin complex associated with axonal transport. The p150^glued subunit plays an essential role in microtubule binding.^14,15) However, the mechanisms underlying the symptoms of Perry disease caused by DCTN1 mutations remain unclear.

Histological analyses have revealed the presence of transactive response DNA-binding protein of 43 kDa (TDP-43)- and dynactin-double-positive aggregates in the neurons of the basal ganglia and brainstem of patients with Perry disease.¹⁴⁾ Many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with TDP-43 inclusions (FTLD-TDP), have recently been considered “TDP-43 proteinopathies” based on the presence of TDP-43 inclusions in postmortem tissues.^16,17) Furthermore, many studies have demonstrated a strong association of protein aggregates in the brain, including amyloid β, phosphorylated tau, and synuclein, with Alzheimer’s and Parkinson’s disease.^18,19) Therefore, protein aggregates play a critical role in the pathogenesis of various neurodegenerative diseases. Further, Araki et al. reported that several p150^glued mutants (F52L, G59S, and G71R) show diffuse cytoplasmic distribution, including intracellular aggregates, whereas the wild-type p150^glued shows filamentous distribution due to microtubule binding without aggregates.⁵⁾ DCTN1 mutation has been widely screened in Perry disease since its discovery in 2009. Notably, 12 distinct DCTN1 mutations in Perry disease have been identified globally in >18 families.³⁾ In contrast, DCTN1 missense mutations have been reported in patients with various other neurodegenerative diseases, such as distal hereditary motor neuropathies, motor neuron disease (MND),^20–22) ALS,²³⁾ FTLD, and progressive supranuclear palsy and in healthy controls.²⁴⁾ Although some DCTN1 missense mutations result in mislocalization of p150^glued and protein aggregation, whether this cellular phenotype is common to all DCTN1 mutations remains unclear. Moreover, Perry disease is an autosomal dominant genetic disorder; therefore, DCTN1 mutants coexist with wild-type proteins under physiological conditions. Nevertheless, whether the presence of wild-type p150^glued can mitigate or enhance the aggregation of the mutant p150^glued remains unclear.

In this study, we comprehensively investigated the cellular phenotypes of p150^glued mutants and assessed the effect of co-overexpressing wild-type p150^glued with the mutants on the formation of mutant aggregates.

MATERIALS AND METHODS

Vector Construction

To construct pCAG–ALFA–DCTN1 (wild-type (WT)), the DCTN1 fragment from human cDNA prepared from HEK293T cells was amplified using PCR, and the ALFA-tag sequence²⁵⁾ was inserted at the N-terminus. Next, the fragment was ligated to a vector backbone prepared from pCAG–EGxxFP (Addgene #50716)²⁶⁾ using NEBuilder HiFi DNA Assembly Master Mix (E2621, New England Biolabs, Ipswich, MA, U.S.A.). To construct pCAG–ALFA–DCTN1(F52L, K56R, G59S, G67D, G71A, G71E, G71R, T72P, Q74P, Y78C, I87T, R1049Q, and R1101K), the mutant DCTN1 fragments from the wildtype fragment were amplified using PCR with mutated primers and ligated with CAG fragment from pCAG–EGxxFP using NEbuilder Assembly Master Mix.

To construct pCAG–GFP–DCTN1(WT), DCTN1 fragments from human cDNA prepared from HEK293T cells were amplified using PCR. In addition, the green fluorescent protein (GFP) fragment was amplified using PCR from the pAAV–CMV–EmGFP–miRNA backbone,²⁷⁾ and the GS-linker sequence (GGGGS)²⁾ was inserted at the N-terminus. These fragments were then ligated to the CAG fragment from pCAG–EGxxFP using the HiFi DNA Assembly Master Mix.

Heterologous Expression of p150^glued-ALFA Overexpression in HEK293T Cells

HEK293T cells (Lenti-X 293T cells, 632180, Clontech, Mountain View, CA, U.S.A.) were grown on poly-L-lysine (PLL)-coated cover glass (Matsunami Glass, Osaka, Japan) in a 24-well dish to 50–60% confluence. Then 0.8 µg of the DCTN1 plasmid (pCAG–ALFA–DCTN1(WT, F52L, K56R, G59S, G67D, G71A, G71E, G71R, T72P, Q74P, Y78C, I87T, R1049Q, and R1101K)), and 1.6 µL of lipofectamine 2000 (Thermo Fisher Scientific, Carlsbad, CA, U.S.A.) were added into 100 µL of Dulbecco’s modified Eagle medium (DMEM). After 15 min incubation, the plasmid mixture was applied to the dish and incubated at 37 °C. Analysis was performed 24 h after transfection. The details of immunocytochemical analysis were described in Supplementary Materials.

Heterologous Expression of p150^glued –GFP and p150^glued –ALFA Co-overexpression in HEK293T Cells

HEK293T cells were grown on PLL-coated glass discs in a 24-well dish to 50–60% confluence. Then 0.4 µg of the ALFA-tagged DCTN1 plasmid (pCAG–ALFA–DCTN1(WT, F52L, K56R, G59S, G67D, G71A, G71E, G71R, T72P, Q74P, Y78C, I87T, R1049Q, and R1101K)), 0.4 µg of the GFP-tagged DCTN1 plasmid (pCAG–GFP–DCTN1(WT)), and 1.6 µL of lipofectamine 2000 (Thermo Fischer Scientific) were added into 100 µL of DMEM. After 15 min incubation, the plasmid mixture was applied to the dish and incubated at 37 °C. Analysis was performed 24 h after transfection.

Statistical Analysis

All data were expressed as means ± standard error of the mean (S.E.M.). Statistical analyses were performed using one-way ANOVA, followed by Tukey’s post-hoc test using GraphPad Prism 10 (GraphPad, San Diego, CA, U.S.A.). In all cases, p < 0.05 was considered statistically significant.

RESULTS

Intracellular Distribution of Wild-Type and Mutant p150^glued

Pathological reports have revealed severe neuronal loss in the substantia nigra and locus coeruleus, and dynactin-positive aggregates in residual neurons in Perry disease.^4,28) To recapitulate the pathology of Perry disease, we investigated the cellular distribution of heterologously expressed wild-type and mutant p150^glued (Fig. 1A). In addition to the mutations previously found in patients with Perry disease, we investigated missense mutations associated with other neurodegenerative disorders and those found in healthy controls. A G59S mutation in DCTN1, characterized by vocal cord paralysis, that has been previously identified in familial motor neuron disease,²⁰⁾ is not observed in Perry disease,²¹⁾ The G59S mutation in p150^glued results in reduced microtubule binding and intracellular aggregation.²²⁾ The R1049Q and R1101K mutations of DCTN1 were found through a large-scale genetic screening of sporadic patients with ALS,²³⁾ and the missense mutation I87T was identified in healthy controls.²⁴⁾ Although previous reports have shown that overexpression of G59S mutant induced aggregate formation in neuronal cell lines,^22,29) SH-SY5Y cells and MN1 cells, we used HEK293T cells in this study according to the many previous reports, which enables us to directly compare our results with previous reports. We evaluated the DCTN1 aggregation 24 h after transfection because a previous report indicates that overexpression of WT DCTN1 induced apoptosis in 48 h but not in 24 h after transfection.²⁹⁾ Immunocytochemical analysis revealed that ALFA-tagged wild-type p150^glued showed a thread-like cytoplasmic distribution, consistent with previous reports^5,14,23) (Fig. 1B). Therefore, the addition of the ALFA-tag to p150^glued did not affect protein localization. Similar to the wild-type, ALFA-tagged K56R, I87T, R1049Q, and R1101K mutants showed a thread-like cytoplasmic distribution (Fig. 1B). In contrast, the mutant p150^glued associated with Perry disease mutations (F52L, G67D, G71R, G71A, G71E, T72P, Q74P, and Y78C), and the G59S mutation found in MND showed diffuse cytoplasmic distribution with aggregates of various sizes (Figs. 1B, C). We found that F52L mutant showed thread-like cytoplasmic distribution in addition to the intracellular aggregates which were relatively small compared to other DCTN1 mutants (Fig. 1C). Moreover, aggregates of F52L mutant tended to show uniform expression pattern in cells, which were drastically different from other DCTN1 mutants (Fig. 1C). Quantitative analysis revealed a significantly higher proportion of cells bearing p150^glued-positive aggregates in transfectants expressing the mutants than that in the wild-type. (F_13,32 = 53.5, p < 0.001, one-way ANOVA; p < 0.001, Tukey’s multiple comparison test; Fig. 1D).

Fig. 1. Intracellular Distribution of Wild-Type and Mutant p150^glued

(A) Schematic of the p150^glued subunit of dynactin. CAP-Gly; microtube binding domain. (B, C) HEK293T cells transfected with ALFA-tagged wild-type (WT) and mutant p150^glued, which is not associated with Perry disease (B), and Perry disease mutant p150^glued (C) were immunostained for p150^glued. Scale bar: 10 µm. PD, Parkinson’s disease; MND, motor neuron disease; HC, healthy control; ALS, amyotrophic lateral sclerosis. (D) Percentage of p150^glued-positive cells containing aggregates. *** p < 0.001 vs. WT, using ordinary one-way ANOVA, post hoc Tukey’s multiple comparison test, n = 3–5, biological replicates in two independent experiments. Values are presented as mean ± S.E.M.

Co-overexpressing Wild-Type p150^glued with Mutants Suppresses Mutant DCTN1 Aggregation

Perry disease is an autosomal dominant genetic disorder; therefore, DCTN1 mutants coexist with wild-type proteins under physiological conditions. To investigate whether the mutants disrupted the distribution of wild-type proteins under physiological conditions, GFP-tagged wild-type p150^glued and ALFA-tagged mutant p150^glued were co-overexpressed heterologously. Immunocytochemical analysis revealed a thread-like cytoplasmic distribution of the ALFA-tagged wild-type p150^glued and K56R, I87T, R1049Q, and R1101K mutants, similar to that in the absence of GFP-tagged WT p150^glued (Fig. 2A). The mutant p150^glued associated with Perry disease (F52L, G67D, G71R, G71A, G71E, T72P, Q74P, and Y78C) showed a thread-like cytoplasmic distribution with fewer p150^glued aggregates, which was strikingly different from the distribution in the absence of GFP-tagged wild-type p150^glued (Fig. 2B). In contrast, the G59S mutant formed cytoplasmic aggregates even in the presence of wild-type p150^glued (Fig. 2A). Quantitative analysis revealed a significantly higher proportion of GFP- and ALFA-double positive cells, including those with ALFA-positive aggregates in transfectants expressing all Perry disease mutants except for the F52L and G59S mutants, than that of the wild type (F_13,28 = 45.7, p < 0.001, one-way ANOVA; p < 0.01 (G67D, G71A, and T72P), p < 0.001 (G59S, G71E, Q74P, G71R, and Y78C), Tukey’s multiple comparison test; Fig. 2C).

Fig. 2. Co-overexpressing Wild-Type p150^glued with Mutants Suppresses Mutant DCTN1 Aggregation

(A, B) HEK293T cells transfected with GFP-tagged wild-type (WT) and ALFA-tagged mutant p150^glued, which is not associated with Perry disease (A), and Perry disease mutant p150^glued (B) were immunostained for GFP or ALFA. Scale bar: 10 µm. (C) Percentage of GFP- and ALFA-double-positive cells containing ALFA-positive aggregates. ** p < 0.01, *** p < 0.001, *** p < 0.001 vs. WT, using ordinary one-way ANOVA, post hoc Tukey’s multiple comparison test, n = 3, biological replicates in two independent experiments. Values are presented as mean ± S.E.M.

DISCUSSION

Our findings revealed that the overexpression of Perry disease mutants led to the formation of intracellular p150^glued aggregates, which was mitigated by wild-type p150^glued. In contrast, mutant p150^glued found in patients with ALS and healthy controls showed a thread-like intracellular distribution similar to that of wild-type p150^glued. Therefore, intracellular p150^glued aggregate formation plays an important role in the pathogenesis of Perry disease.

Notably, among all Perry disease mutants, the F52L mutant produced fewer aggregates. We found that F52L-expressing cells contain thread-like expression of p150^glued in the cytoplasm compared to other mutants (Fig. 1C), indicating that microtubule binding capability of F52L may be partly conserved compared to other mutants. Indeed, Araki et al. reported that microtubule binding capability of F52L mutant is around 80% of that of WT while G59S and G71R mutants show microtubule binding capability of less than 60% of WT,⁵⁾ supporting this possibility, although exact mechanism underlying the weak tendency of F52L mutant to form aggregates should be further investigated. A patient bearing this mutation from Japan showed relatively slow disease progression, and the disease onset occurred at the age of 48–70 years.⁵⁾ Therefore, slow disease progression may be due to the weak tendency of the F52L mutant to produce intracellular aggregates. Further, differences in the distribution among the mutants that formed aggregates were observed when they were co-expressed with the wild-type protein, depending on whether they were associated with Perry disease. Neurodegeneration in the basal ganglia, brainstem, and upper motor neurons is observed in patients with Perry disease.³⁾ In contrast, patients with the G59S mutation exhibit neurodegeneration only in lower motor neurons.²¹⁾ Notably, in our study, the G59S mutant showed more robust aggregate-forming capability than the mutants associated with Perry disease, which was frequently observed in wild-type p150^glued. Consistent with these results, disease onset in patients with the G59S mutation was at the age of 30 years, which is much earlier than that of Perry disease (49 years),²⁰⁾ supporting the hypothesis that the strength of intracellular aggregate formation is linked to earlier disease onset.

As p150^glued forms dimers through the interaction between coiled-coil domains to exert its function,³⁰⁾ dimerization of the wild-type and mutant proteins may occur when expressed simultaneously. The suppression of mutant protein aggregate formation upon co-expression with the wild-type protein suggests that the wild-type protein may actively interact with the mutant protein, potentially preventing mislocalization of the p150^glued mutants. Furthermore, we rarely found WT DCTN1 in aggregates of the DCTN1 mutants although some co-aggregates were found as shown in Fig. 2B (Q74P). One possible explanation is that aggregates are formed by dimer of DCTN1 mutants, and hetero dimer of WT and mutant DCTN1 can bind to microtubule and thus does not form aggregate. In contrast, the G59S mutation induces functional impairment through a change in the folding structure,²⁰⁾ suggesting that the wild-type protein may not form dimers or suppress G59S mutant aggregation (Fig. 2A).

Homozygous Dctn1 knock-out mice are embryonically lethal.³¹⁾ However, heterozygous Dctn1 knock-out mice did not develop the neuropathological abnormalities observed in heterozygous Dctn1^G59S knock-in mice.³¹⁾ Therefore, Perry disease may occur due to the toxic function of DCTN1 mutant but not due to haploinsufficiency.³¹⁾ In this study, co-expression of wild-type and mutant p150^glued showed seemingly normal cellular localization. However, further investigation is needed to evaluate whether this suppression of the mislocalization of mutants leads to functional normalization.

The mechanism underlying the intracellular aggregate formation in Perry disease remains undetermined. Our findings revealed that the re-localization of mutant p150^glued by wild-type p150^glued expression mitigated aggregate formation, indicating that reduced binding to endogenous targets and microtubules may trigger aggregate formation. Interestingly, a recent study indicated that TDP-43, an RNA-binding protein, forms intranuclear and cytoplasmic aggregates through reduced RNA binding and a subsequent decrease in HSP70 binding,²⁹⁾ supporting this possibility. Moreover, mutant p150^glued aggregate content remains uninvestigated. Cytoplasmic aggregates of p150^glued do not colocalize with TDP-43 in mutant p150^glued overexpressing cells.^29,33) In contrast, intracellular aggregates containing p150^glued and ubiquitin have been found in dopamine neurons differentiated from the induced pluripotent stem cells of a patient with Perry disease.³³⁾ Therefore, mutant p150^glued aggregates may contain ubiquitinated proteins, which are degraded in the proteasome under physiological conditions. The detailed structures of protein aggregates found in neurodegenerative disorders, including ALS, Alzheimer’s disease, and Parkinson’s disease,^34–36) were identified using cryo-electron microscopy. Analysis of protein aggregates from patients with Perry disease and Perry disease-specific induced pluripotent stem cells may provide deeper insights into the biological basis and drug development for Perry disease.

The limitation of this study is two-fold. In this study, we used potent CAG promoter comparable to another potent CMV promoter used in the previous report.²²⁾ However, it is important to investigate whether DCTN1 aggregation is induced by moderate expression of the mutants considering moderate to potent expression level of dynactin in the brain.³⁰⁾ Second limitation is that we did not observe cell death 24 h after transfection in this study. Although this result is consistent to previous report,²⁹⁾ further analysis in different time points and neuronal cells will help understand the mechanism whether and how intracellular DCTN1 aggregates result in neurodegeneration observed in the patients with Perry disease.²⁸⁾

In conclusion, the overexpression of Perry disease mutants led to the formation of intracellular p150^glued aggregates, which was suppressed by wild-type p150^glued. In contrast, mutant p150^glued found in patients with ALS and healthy controls showed a thread-like intracellular distribution similar to that of wild-type p150^glued. Further investigation of the mechanism of aggregate formation, contents of the aggregates, and biological mechanisms of Perry disease may help develop novel therapeutics.

Acknowledgments

We thank Dr. M. Ikawa (pCAG–EGxxFP; #50716; Addgene) for providing the construct. We thank Dr. T. Mishima (Fukuoka University) for critical advice on the immunocytochemical analysis of p150^glued. This work was supported by JST FOREST, Grant number JPMJFR2268 (K.N.); JSPS KAKENHI, Grant numbers JP20H04774 (K.N.), JP20K07064 (K.N.), JP18H04616 (S.K.), and JP20H00491 (S.K.); AMED, Grant numbers JP20ak0101088h0003 (S.K.) and JP21ak0101153h0001 (S.K.), and Grants from the Suzuken Memorial Foundation (K.N.), the Lotte Research Promotion Grant (K.N.), and the Takeda Science Foundation (K.N.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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