Compartmentalized citrullination in Muller glial endfeet during retinal degeneration

Palko, Sarah Ilona; Saba, N.; Mullane, Elias; Nicholas, Benjamin D.; Nagasaka, Yosuke; Ambati, Jayakrishna; Gelfand, Bradley D.; Ishigami, Akihito; Bargagna‐Mohan, Paola; Mohan, Royce

doi:10.1073/pnas.2121875119

Cited by 17 publications

(41 citation statements)

References 14 publications

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“…Our results show that PC is dynamically altered during disease progression in these mouse models. Whereas PC and PAD2 expression are generally increased in reactive astrocytes as reported in studies of other neurodegenerative disease models [20,23,[39][40][41], our study has unveiled new, previously unknown changes. We demonstrate that, in contrast to the increase in astrocytes, PC and PAD2 expression trend downwards in neurons as disease progresses (Figs.…”

Section: Discussionsupporting

confidence: 75%

Protein citrullination marks myelin protein aggregation and disease progression in mouse ALS models

Yusuf

Qiao

Parsi

et al. 2022

acta neuropathol commun

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Increased protein citrullination (PC) and dysregulated protein arginine deiminase (PAD) activity have been observed in several neurodegenerative diseases. PC is a posttranslational modification catalyzed by the PADs. PC converts peptidyl-arginine to peptidyl-citrulline, thereby reducing the positive charges and altering structure and function of proteins. Of the five PADs, PAD2 is the dominant isoform in the central nervous system (CNS). Abnormal PC and PAD dysregulation are associated with numerous pathological conditions, including inflammatory diseases and neurodegeneration. Animal model studies have shown therapeutic efficacy from inhibition of PADs, thus suggesting a role of PC in pathogenesis. To determine whether PC contribute to amyotrophic lateral sclerosis (ALS), a deadly neurodegenerative disease characterized by loss of motor neurons, paralysis, and eventual death, we investigated alterations of PC and PAD2 in two different transgenic mouse models of ALS expressing human mutant SOD1G93A and PFN1C71G, respectively. PC and PAD2 expression are altered dynamically in the spinal cord during disease progression in both models. PC and PAD2 increase progressively in astrocytes with the development of reactive astrogliosis, while decreasing in neurons. Importantly, in the spinal cord white matter, PC accumulates in protein aggregates that contain the myelin proteins PLP and MBP. PC also accumulates progressively in insoluble protein fractions during disease progression. Finally, increased PC and PAD2 expression spatially correlate with areas of the CNS with the most severe motor neuron degeneration. These results suggest that altered PC is an integral part of the neurodegenerative process and potential biomarkers for disease progression in ALS. Moreover, increased PC may contribute to disease-associated processes such as myelin protein aggregation, myelin degeneration, and astrogliosis.

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Section: Discussionsupporting

confidence: 75%

Protein citrullination marks myelin protein aggregation and disease progression in mouse ALS models

Yusuf

Qiao

Parsi

et al. 2022

acta neuropathol commun

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“…PAD4 colocalization on filamentous GFAP was also observed in the murine laser-induced model of CNV (Palko et al, 2022). Defining PAD4's key role in this PTM, conditional glial-specific PAD4 knockout (PAD4cKO) mice subjected to laser injury showed significantly reduced hypercitrullination (Palko et al, 2022).…”

Section: Introductionmentioning

confidence: 91%

“…However, whether such a mechanism occurs in genetic diseases was subsequently revealed in a genetic model of spontaneous retinal degeneration (JR5558 mice) that revealed heightened citrullination (hypercitrullination), with both citrullinated GFAP and PAD4 being localized in reactive Müller glia. Such maladaptive chronic retinal hypercitrullination was also seen in severely diseased human wet-AMD maculae demonstrating that hypercitrullination is retained into end stages of retinal disease (Palko et al, 2022). Because Müller glial endfeet play such a critical role in pathological hypercitrullination, we have termed it the citrullination bunker (Palko et al, 2022).…”

Section: Introductionmentioning

confidence: 99%

“…Such maladaptive chronic retinal hypercitrullination was also seen in severely diseased human wet-AMD maculae demonstrating that hypercitrullination is retained into end stages of retinal disease (Palko et al, 2022). Because Müller glial endfeet play such a critical role in pathological hypercitrullination, we have termed it the citrullination bunker (Palko et al, 2022).…”

Section: Introductionmentioning

confidence: 99%

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Peptidyl arginine deiminase 4 deficiency protects against subretinal fibrosis by inhibiting Müller glial hypercitrullination

Palko

Saba

Bargagna‐Mohan

et al. 2022

J of Neuroscience Research

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Retinal scarring with vision loss continues to be an enigma in individuals with advanced age‐related macular degeneration (AMD). Müller glial cells are believed to initiate and perpetuate scarring in retinal degeneration as these glial cells participate in reactive gliosis and undergo hypertrophy. We previously showed in the murine laser‐induced model of choroidal neovascularization that models wet‐AMD that glial fibrillary acidic protein (GFAP) expression, an early marker of reactive gliosis, increases along with its posttranslational modification citrullination. This was related to increased co‐expression of the citrullination enzyme peptidyl arginine deiminase‐4 (PAD4), which also colocalizes to GFAP filaments. However, whether such hypercitrullination in Müller glial drives fibrotic pathology has remained understudied. Here, using male and female C57Bl6 mice subjected to laser injury, we investigated in a temporal study how citrullination impacts GFAP and PAD4 dynamics. We found that high molecular weight citrullinated species that accumulate in Müller glia corresponded with dynamic changes in GFAP and PAD4 showing their temporal redistribution from polymeric cytoskeletal to soluble protein fractions using immunostaining and western blot analysis. In conditional glial‐specific PAD4 knockout (PAD4cKO) mice subjected to laser injury, there was a stark reduction of citrullination and of polymerized GFAP filaments. These injured PAD4cKO retinas showed improved lesion healing, as well as reduced fibronectin deposition in the subretinal space at 30 days. Taken together, these findings reveal that pathologically overexpressed PAD4 in reactive Müller glia governs GFAP filament dynamics and alters their stability, suggesting chronic PAD4‐driven hypercitrullination may be a target for retinal fibrosis.

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“…In addition, the expression of several Müller glia-specific genes was altered by reserpine, with GFAP levels reduced in both organoids and mouse retina in vivo . Müller glia are believed to play a major role in reactive gliosis and likely adapt their transcriptome to support photoreceptors in retinal degeneration ( 95, 96 ). Yet, we are unsure whether reserpine directly acts on Müller glia to augment photoreceptor survival, or its response is a consequence of improved microenvironment and reduced retinal stress.…”

Section: Discussionmentioning

confidence: 99%

Reserpine maintains photoreceptor survival in retinal ciliopathy by resolving proteostasis imbalance and ciliogenesis defects

Chen

Swaroop

Papal

et al. 2022

Preprint

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Ciliopathies manifest from sensory abnormalities to syndromic disorders with multiorgan pathologies, with retinal degeneration a highly penetrant phenotype. Photoreceptor cell death is a major cause of incurable blindness in retinal ciliopathies. To identify drug candidates to maintain photoreceptor survival, we performed an unbiased, high-throughput screening of over 6,000 bioactive small molecules using retinal organoids differentiated from induced pluripotent stem cells (iPSC) of rd16 mouse, which is a model of Leber congenital amaurosis (LCA)10 caused by mutations in the cilia-centrosomal gene CEP290. We identified five non-toxic positive hits, including the lead molecule reserpine, which improved photoreceptor survival in rd16 organoids. Reserpine also maintained photoreceptors in retinal organoids derived from induced pluripotent stem cells of LCA10 patients and in rd16 mouse retina in vivo. Reserpine-treated patient organoids revealed modulation of signaling pathways related to cell survival/death, metabolism, and proteostasis. Further investigation uncovered misregulation of autophagy associated with compromised primary cilium biogenesis in patient organoids and rd16 mouse retina. Reserpine partially restored the balance between autophagy and the ubiquitin-proteasome system, at least in part by increasing the cargo adaptor p62 and improving primary cilium assembly. Our study identifies effective drug candidates in preclinical studies of CEP290 retinal ciliopathies through cross-species drug discovery using iPSC-derived organoids, highlights the impact of proteostasis in the pathogenesis of ciliopathies, and provides new insights for treatments of retinal neurodegeneration.

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Compartmentalized citrullination in Muller glial endfeet during retinal degeneration

Cited by 17 publications

References 14 publications

Protein citrullination marks myelin protein aggregation and disease progression in mouse ALS models

Protein citrullination marks myelin protein aggregation and disease progression in mouse ALS models

Peptidyl arginine deiminase 4 deficiency protects against subretinal fibrosis by inhibiting Müller glial hypercitrullination

Reserpine maintains photoreceptor survival in retinal ciliopathy by resolving proteostasis imbalance and ciliogenesis defects

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