Identification and cellular localization in Xenopus laevis photoreceptors of three Peripherin-2 family members, Prph2, Rom1 and Gp2l, which arose from gene duplication events in the common ancestors of jawed vertebrates.

Rod and cone photoreceptors are named for the distinct morphologies of their outer segment organelles, which are either cylindrical or conical, respectively. The morphologies of the stacked disks that comprise the rod and cone outer segments also differ: rod disks are completely sealed and are discontinuous from the plasma membrane, while cone disks remain partially open to the extracellular space. These morphological differences between photoreceptor types are more prominent in non-mammalian vertebrates, whose cones typically possess a greater proportion of open disks and are more tapered in shape. In mammals, the tetraspanin prph2 generates and maintains the highly curved disk rim regions by forming extended oligomeric structures with itself and a structurally similar paralog, rom1. Here we determined that in addition to these two proteins, there is a third Prph2 family paralog in most non-mammalian vertebrate species, including X. laevis: Glycoprotein 2-like protein or "Gp2l". A survey of multiple genome databases revealed a single invertebrate Prph2 'pro-ortholog' in Amphioxus, several echinoderms and in a diversity of protostomes indicating an ancient divergence from other tetraspanins. Based on phylogenetic analysis, duplication of the vertebrate predecessor likely gave rise to the Gp2l and Prph2/Rom1 clades, with a further duplication distinguishing the Prph2 and Rom1 clades. Mammals have lost Gp2l and their Rom1 has undergone a period of accelerated evolution such that it has lost several features that are retained in non-mammalian vertebrate Rom1. Specifically, Prph2, Gp2l and non-mammalian Rom1 encode proteins with consensus N-linked glycosylation and outer segment localization signals; mammalian rom1 lacks these motifs. We determined that X. laevis gp2l is expressed exclusively in cones and green rods, while X. laevis rom1 is expressed exclusively in rods, and prph2 is present in both rods and cones. The presence of three Prph2-related genes with distinct expression patterns as well as the rapid evolution of mammalian Rom1, may contribute to the more pronounced differences in morphology between rod and cone outer segments and rod and cone disks observed in non-mammalian versus mammalian vertebrates.

Peripherin is a biomarker of axonal damage in peripheral nervous system disease.

Valid, responsive blood biomarkers specific to peripheral nerve damage would improve management of peripheral nervous system (PNS) diseases. Neurofilament light chain (NfL) is sensitive for detecting axonal pathology but is not specific to PNS damage, as it is expressed throughout the PNS and CNS. Peripherin, another intermediate filament protein, is almost exclusively expressed in peripheral nerve axons. We postulated that peripherin would be a promising blood biomarker of PNS axonal damage. We demonstrated that peripherin is distributed in sciatic nerve, and to a lesser extent spinal cord tissue lysates, but not in brain or extra-neural tissues. In the spinal cord, anti-peripherin antibody bound only to the primary cells of the periphery (anterior horn cells, motor axons and primary afferent sensory axons). In vitro models of antibody-mediated axonal and demyelinating nerve injury showed marked elevation of peripherin levels only in axonal damage and only a minimal rise in demyelination. We developed an immunoassay using single molecule array technology for the detection of serum peripherin as a biomarker for PNS axonal damage. We examined longitudinal serum peripherin and NfL concentrations in individuals with Guillain-Barré syndrome (GBS, n = 45, 179 time points), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP, n = 35, 70 time points), multiple sclerosis (n = 30), dementia (as non-inflammatory CNS controls, n = 30) and healthy individuals (n = 24). Peak peripherin levels were higher in GBS than all other groups (median 18.75 pg/ml versus < 6.98 pg/ml, P < 0.0001). Peak NfL was highest in GBS (median 220.8 pg/ml) and lowest in healthy controls (median 5.6 pg/ml), but NfL did not distinguish between CIDP (17.3 pg/ml), multiple sclerosis (21.5 pg/ml) and dementia (29.9 pg/ml). While peak NfL levels were higher with older age (rho = +0.39, P < 0.0001), peak peripherin levels did not vary with age. In GBS, local regression analysis of serial peripherin in the majority of individuals with three or more time points of data (16/25) displayed a rise-and-fall pattern with the highest value within the first week of initial assessment. Similar analysis of serial NfL concentrations showed a later peak at 16 days. Group analysis of serum peripherin and NfL levels in GBS and CIDP patients were not significantly associated with clinical data, but in some individuals with GBS, peripherin levels appeared to better reflect clinical outcome measure improvement. Serum peripherin is a promising new, dynamic and specific biomarker of acute PNS axonal damage.

Peripherin: A proposed biomarker of traumatic axonal injury triggered by mechanical force.

Traumatic axonal injury (TAI) is one of the most common pathological features of severe traumatic brain injury (TBI). Our previous study using proteomics suggested that peripherin (PRPH) should be a potential candidate as a biomarker for TAI diagnosis. This study is to further elucidate the role and association of PRPH with TAI. In the animal study, we performed immunohistochemistry, ELISA and morphological analysis to evaluate PRPH level and distribution following a severe impact. PRPH-positive regions were widely distributed in the axonal tract throughout the whole brain. Axonal injuries with PRPH inclusion were observed post-TBI. Besides, PRPH was significantly increased in both cerebral spinal fluid and plasma at the early phase post-TBI. Colocalization analysis based on microscopy revealed that PRPH represents an immunohistological biomarker in the neuropathological diagnosis of TAI. Brain samples from patients with TBI were included to further test whether PRPH is feasible in the real practice of neuropathology. Immunohistochemistry of PRPH, NFH, APP and NFL on human brain tissues further confirmed PRPH as an immunohistological biomarker that could be applied in practice. Collectively, we conclude that PRPH mirrors the cytoskeleton injury of axons and could represent a neuropathological biomarker for TAI.

Prph2 disease mutations lead to structural and functional defects in the RPE.

Prph2 is a photoreceptor-specific tetraspanin with an essential role in the structure and function of photoreceptor outer segments. PRPH2 mutations cause a multitude of retinal diseases characterized by the degeneration of photoreceptors as well as defects in neighboring tissues such as the RPE. While extensive research has analyzed photoreceptors, less attention has been paid to these secondary defects. Here, we use different Prph2 disease models to evaluate the damage of the RPE arising from photoreceptor defects. In Prph2 disease models, the RPE exhibits structural abnormalities and cell loss. Furthermore, RPE functional defects are observed, including impaired clearance of phagocytosed outer segment material and increased microglia activation. The severity of RPE damage is different between models, suggesting that the different abnormal outer segment structures caused by Prph2 disease mutations lead to varying degrees of RPE stress and thus influence the clinical phenotype observed in patients.

Enterovirus-A71 exploits peripherin and Rac1 to invade the central nervous system.

Enterovirus-A71 (EV-A71) has been associated with severe neurological forms of hand, foot, and mouth disease (HFMD). EV-A71 infects motor neurons at neuromuscular junctions (NMJs) to invade the central nervous system (CNS). Here, we investigate the role of peripherin (PRPH) during EV-A71 infection, a type III intermediate neurofilament involved in neurodegenerative conditions. In mice infected with EV-A71, PRPH co-localizes with viral particles in the muscles at NMJs and in the spinal cord. In motor neuron-like and neuroblastoma cell lines, surface-expressed PRPH facilitates viral entry, while intracellular PRPH influences viral genome replication through interactions with structural and non-structural viral components. Importantly, PRPH does not play a role during infection with coxsackievirus A16, another causative agent of HFMD rarely associated with neurological complications, suggesting that EV-A71 ability to exploit PRPH represents a unique attribute for successful CNS invasion. Finally, we show that EV-A71 also exploits some of the many PRPH-interacting partners. Of these, small GTP-binding protein Rac1 represents a potential druggable host target to limit neuroinvasion of EV-A71.

The Role of Peripherin-2/ROM1 Complexes in Photoreceptor Outer Segment Disc Morphogenesis.

The light-sensitive outer segment organelle of photoreceptor cells contains a stack of hundreds of flat, disc-shaped membranes called discs. The rims of these discs contain a photoreceptor-specific tetraspanin protein peripherin-2 (also known as rds or PRPH2). Mutations in the PRPH2 gene lead to a wide variety of inherited retinal degenerations in humans. The vast majority of these mutations occur within a large, intradiscal loop of peripherin-2, known as the D2 loop. The D2 loop mediates well-established intermolecular interactions of peripherin-2 molecules among themselves and a homologous protein ROM1. These interactions lead to the formation of large, highly ordered oligomers. In this chapter, we discuss the supramolecular organization of peripherin-2/ROM1 complexes and their contribution to the process of outer segment disc morphogenesis and enclosure.

Prime Editing Strategy to Install the PRPH2 c.828+1G>A Mutation.

Mutations in peripherin 2 (PRPH2) are associated with a spectrum of inherited retinal diseases (IRDs) including retinitis pigmentosa (RP) and macular degeneration. As PRPH2 is localized to cone and rod outer segments, mutations in PRPH2 lead the disorganization or absence of photoreceptor outer segments. Here, we report on a patient with PRPH2-linked RP who exhibited widespread RPE atrophy with a central area of macular atrophy sparing the fovea. In future studies, we plan to model the pathobiology of PRPH2-based RP using induced pluripotent stem cell (iPSC)-derived retinal organoids. To effectively model rare mutations using iPSC-derived retinal organoids, we first require a strategy that can install the desired mutation in healthy wild-type iPSC, which can efficiently generate well-laminated retinal organoids. In this study, we developed an efficient prime editing strategy for the installation of the pathogenic PRPH2 c.828+1 G>A splice-site mutation underlying our patient's disease.

Multistep peripherin-2/rds self-assembly drives membrane curvature for outer segment disk architecture and photoreceptor viability.

Rod and cone photoreceptor outer segment (OS) structural integrity is essential for normal vision; disruptions contribute to a broad variety of retinal ciliopathies. OSs possess many hundreds of stacked membranous disks, which capture photons and scaffold the phototransduction cascade. Although the molecular basis of OS structure remains unresolved, recent studies suggest that the photoreceptor-specific tetraspanin, peripherin-2/rds (P/rds), may contribute to the highly curved rim domains at disk edges. Here, we demonstrate that tetrameric P/rds self-assembly is required for generating high-curvature membranes in cellulo, implicating the noncovalent tetramer as a minimal unit of function. P/rds activity was promoted by disulfide-mediated tetramer polymerization, which transformed localized regions of curvature into high-curvature tubules of extended lengths. Transmission electron microscopy visualization of P/rds purified from OS membranes revealed disulfide-linked tetramer chains up to 100 nm long, suggesting that chains maintain membrane curvature continuity over extended distances. We tested this idea in Xenopus laevis photoreceptors, and found that transgenic expression of nonchain-forming P/rds generated abundant high-curvature OS membranes, which were improperly but specifically organized as ectopic incisures and disk rims. These striking phenotypes demonstrate the importance of P/rds tetramer chain formation for the continuity of rim formation during disk morphogenesis. Overall, this study advances understanding of the normal structure and function of P/rds for OS architecture and biogenesis, and clarifies how pathogenic loss-of-function mutations in P/rds cause photoreceptor structural defects to trigger progressive retinal degenerations. It also introduces the possibility that other tetraspanins may generate or sense membrane curvature in support of diverse biological functions.

Syntaxin 3 is essential for photoreceptor outer segment protein trafficking and survival.

Trafficking of photoreceptor membrane proteins from their site of synthesis in the inner segment (IS) to the outer segment (OS) is critical for photoreceptor function and vision. Here we evaluate the role of syntaxin 3 (STX3), in trafficking of OS membrane proteins such as peripherin 2 (PRPH2) and rhodopsin. Photoreceptor-specific Stx3 knockouts [Stx3f/f(iCre75) and Stx3f/f(CRX-Cre) ] exhibited rapid, early-onset photoreceptor degeneration and functional decline characterized by structural defects in IS, OS, and synaptic terminals. Critically, in the absence of STX3, OS proteins such as PRPH2, the PRPH2 binding partner, rod outer segment membrane protein 1 (ROM1), and rhodopsin were mislocalized along the microtubules to the IS, cell body, and synaptic region. We find that the PRPH2 C-terminal domain interacts with STX3 as well as other photoreceptor SNAREs, and our findings indicate that STX3 is an essential part of the trafficking pathway for both disc (rhodopsin) and rim (PRPH2/ROM1) components of the OS.

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OBJECTIVES: Nerve growth factor (NGF) induces neuron transdifferentiation of adrenal medulla chromaffin cells (AMCCs) and consequently downregulates the secretion of epinephrine (EPI), which may be involved in the pathogenesis of bronchial asthma. Mammalian achaete scute-homologous 1 (MASH1), a key regulator of neurogenesis in the nervous system, has been proved to be elevated in AMCCs with neuron transdifferentiation in vivo. This study aims to explore the role of MASH1 in the process of neuron transdifferentiation of AMCCs and the mechanisms. METHODS: Rat AMCCs were isolated and cultured. AMCCs were transfected with siMASH1 or MASH1 overexpression plasmid, then were stimulated with NGF and/or dexamethasone, PD98059 (a MAPK kinase-1 inhibitor) for 48 hours. Morphological changes were observed using light and electron microscope. Phenylethanolamine-N-methyltransferase (PNMT, the key enzyme for epinephrine synthesis) and tyrosine hydroxylase were detected by immunofluorescence. Western blotting was used to test the protein levels of PNMT, MASH1, peripherin (neuronal markers), extracellular regulated protein kinases (ERK), phosphorylated extracellular regulated protein kinases (pERK), and JMJD3. Real-time RT-PCR was applied to analyze the mRNA levels of MASH1 and JMJD3. EPI levels in the cellular supernatant were measured using ELISA. RESULTS: Cells with both tyrosine hydroxylase and PNMT positive by immunofluorescence were proved to be AMCCs. Exposure to NGF, AMCCs exhibited neurite-like processes concomitant with increases in pERK/ERK, peripherin, and MASH1 levels (all P<0.05). Additionally, impairment of endocrine phenotype was proved by a signifcant decrease in the PNMT level and the secretion of EPI from AMCCs (all P<0.01). MASH1 interference reversed the effect of NGF, causing increases in the levels of PNMT and EPI, conversely reduced the peripherin level and cell processes (all P<0.01). MASH1 overexpression significantly increased the number of cell processes and peripherin level, while decreased the levels of PNMT and EPI (all P<0.01). Compared with the NGF group, the levels of MASH1, JMJD3 protein and mRNA in AMCCs in the NGF+PD98059 group were decreased (all P<0.05). After treatment with PD98059 and dexamethasone, the effect of NGF on promoting the transdifferentiation of AMCCs was inhibited, and the number of cell processes and EPI levels were decreased (both P<0.05). In addition, the activity of the pERK/MASH1 pathway activated by NGF was also inhibited. CONCLUSIONS: MASH1 is the key factor in neuron transdifferentiation of AMCCs. NGF-induced neuron transdifferentiation is probably mediated via pERK/MASH1 signaling.

Photoreceptor Disc Enclosure Is Tightly Controlled by Peripherin-2 Oligomerization.

Mutations in the PRPH2 gene encoding the photoreceptor-specific protein PRPH2 (also known as peripherin-2 or rds) cause a broad range of autosomal dominant retinal diseases. Most of these mutations affect the structure of the light-sensitive photoreceptor outer segment, which is composed of a stack of flattened "disc" membranes surrounded by the plasma membrane. The outer segment is renewed on a daily basis in a process whereby new discs are added at the outer segment base and old discs are shed at the outer segment tip. New discs are formed as serial membrane evaginations, which eventually enclose through a complex process of membrane remodeling (completely in rods and partially in cones). As disc enclosure proceeds, PRPH2 localizes to the rims of enclosed discs where it forms oligomers which fortify the highly curved membrane structure of these rims. In this study, we analyzed the outer segment phenotypes of mice of both sexes bearing a single copy of either the C150S or the Y141C PRPH2 mutation known to prevent or increase the degree of PRPH2 oligomerization, respectively. Strikingly, both mutations increased the number of newly forming, not-yet-enclosed discs, indicating that the precision of disc enclosure is regulated by PRPH2 oligomerization. Without tightly controlled enclosure, discs occasionally over-elongate and form large membranous "whorls" instead of disc stacks. These data show that the defects in outer segment structure arising from abnormal PRPH2 oligomerization are manifested at the stage of disc enclosure.SIGNIFICANCE STATEMENT The light-sensitive photoreceptor outer segment contains a stack of flattened "disc" membranes that are surrounded, or "enclosed," by the outer segment membrane. Disc enclosure is an adaptation increasing photoreceptor light sensitivity by facilitating the diffusion of the second messenger along the outer segment axes. However, the molecular mechanisms by which photoreceptor discs enclose within the outer segment membrane remain poorly understood. We now demonstrate that oligomers of the photoreceptor-specific protein peripherin-2, or PRPH2, play an active role in this process. We further propose that defects in disc enclosure because of abnormal PRPH2 oligomerization result in major structural abnormalities of the outer segment, ultimately leading to loss of visual function and cell degeneration in PRPH2 mutant models and human patients.

ROM1 contributes to phenotypic heterogeneity in PRPH2-associated retinal disease.

Peripherin 2 (PRPH2) is a retina-specific tetraspanin protein essential for the formation of rod and cone photoreceptor outer segments (OS). Patients with mutations in PRPH2 exhibit severe retinal degeneration characterized by vast inter- and intra-familial phenotypic heterogeneity. To help understand contributors to this within-mutation disease variability, we asked whether the PRPH2 binding partner rod OS membrane protein 1 (ROM1) could serve as a phenotypic modifier. We utilized knockin and transgenic mouse models to evaluate the structural, functional and biochemical effects of eliminating one allele of Rom1 (Rom1+/-) in three different Prph2 models which mimic human disease: C213Y Prph2 (Prph2C/+), K153Del Prph2 (Prph2K/+) and R172W (Prph2R172W). Reducing Rom1 in the absence of Prph2 mutations (Rom1+/-) had no effect on retinal structure or function. However, the effects of reducing Rom1 in the presence of Prph2 mutations were highly variable. Prph2K/+/Rom1+/- mice had improved rod and cone function compared with Prph2K/+ as well as amelioration of K153Del-associated defects in PRPH2/ROM1 oligomerization. In contrast, Prph2R172W/Rom1+/- animals had worsened rod and cone function and exacerbated retinal degeneration compared with Prph2R172W animals. Removing one allele of Rom1 had no effect in Prph2C/+. Combined, our findings support a role for non-pathogenic ROM1 null variants in contributing to phenotypic variability in mutant PRPH2-associated retinal degeneration. Since the effects of Rom1 reduction are variable, our data suggest that this contribution is specific to the type of Prph2 mutation.

PRPF31 interacts with PRPH2 confirmed by co-immunoprecipitation and co-localization.

Both PRPF31 and PRPH2 are the causative genes for retinitis pigmentosa. And both of them are associated with the balance of rhodopsin. In this study, we aim to investigate the co-expression and interaction of PRPF31 and PRPH2. We used PRPF31-eGFP, PRPF31-3xFlag and PRPH2-mCherry vectors were transfected into HEK293T and APRE-19 cells. Immunoblotting and co-immunoprecipitation (Co-IP) were used for gene expression validation and protein interaction. Immunofluorescence staining assay was used to test the co-localization analysis of PRPF31 and PRPH2. Co-IP experiments showed that PRPF31 could be pulled down with an anti-PRPH2 antibody. There was co-localization between PRPF31 and PRPH2 in HEK293T, APRE-19 and mouse retina. The Co-IP and co-localization experiments suggest that PRPF31 interacted with PRPH2.

Integrative study of gene expression datasets in retinal samples of Diabetic Retinopathy.

Diabetic Retinopathy is prevalent among patients with uncontrolled hyperglycemia resulting in vision loss. Despite numerous challenges to create a link among these conditions, the characterization of pathological neovascularization causing retinal damage due to the prognosis of early non-proliferative diabetic retinopathy to late proliferative diabetic retinopathy needs deep understanding. In this study, meta-analysis-based integration of gene expression datasets for the fibrovascular membrane of PDR and neural retina of NPDR were compared, to investigate the differentially expressed genes involved in retinal angiogenesis. Human samples with gene expression profiling of the same experiment type and platform with sufficient information for analysis were included in the study. The studies from cell lines and non-human studies, human samples that include serum, cornea, lens, and/or other ocular tissues or fluids, and studies that lack basic information for analysis were excluded. The microarray datasets available in the Gene Expression Omnibus database of the early and late stages in DR were screened to find common gene expression profiles. Using the INMEX bioinformatics tool, significantly upregulated and downregulated genes in the neural retina of Non-Proliferative Diabetic Retinopathy and fibrovascular membrane of Proliferative Diabetic Retinopathy were compared and studied by the combine effect size method. Using the STRING database PPI network, 50 upregulated and 50 downregulated genes were used to find the key candidate genes involved in retinal disease/degeneration in eye/retinal tissues. In the extensive gene expression meta-analysis performed using INMEX bioinformatics tool, overall, 7935 differentially expressed genes were identified and the respective heatmap was created by using the visualization tools of INVEX. STRING database PPI network identified Retinol Binding Protein 3, Neural Retina Leucine Zipper, S-Antigen Visual Arrestin, Peripherin 2, and Aryl Hydrocarbon Receptor Interacting Protein Like-1 to be the most highly ranked hub genes. The newly discovered potential genes related to retinal angiogenesis causing FVM formation in DR may provide insight into the cellular pathogenesis of NPDR to PDR.

Role of the Intermediate Filament Protein Peripherin in Health and Disease.

Intermediate filaments are the most heterogeneous class among cytoskeletal elements. While some of them have been well-characterized, little is known about peripherin. Peripherin is a class III intermediate filament protein with a specific expression in the peripheral nervous system. Epigenetic modifications are involved in this cell-type-specific expression. Peripherin has important roles in neurite outgrowth and stability, axonal transport, and axonal myelination. Moreover, peripherin interacts with proteins involved in vesicular trafficking, signal transduction, DNA/RNA processing, protein folding, and mitochondrial metabolism, suggesting a role in all these processes. This review collects information regarding peripherin gene regulation, post-translational modifications, and functions and its involvement in the onset of a number of diseases.

Synergic Effect of Early Administration of Probiotics and Adipose-Derived Mesenchymal Stem Cells on Alleviating Inflammation-Induced Chronic Neuropathic Pain in Rodents.

This study investigated the hypothesis that probiotics enhanced the therapeutic effect of adipose-derived mesenchymal stem cells (ADMSCs) on alleviating neuropathic pain (NP) due to chronic constriction injury (CCI) mainly through regulating the microbiota in rats. SD rats (n = 50) were categorized into group 1 (sham-control), group 2 (NP), group 3 (NP + probiotics (i.e., 1.5 billion C.F.U./day/rat, orally 3 h after NP procedure, followed by QOD 30 times)), group 4 (NP + ADMSCs (3.0 × 105 cells) 3 h after CCI procedure, followed by QOD six times (i.e., seven times in total, i.e., mimic a clinical setting of drug use) and group 5 (NP + probiotics + ADMSCs (3.0 × 105 cells)) and euthanized by day 60 after NP induction. By day 28 after NP induction, flow-cytometric analysis showed circulating levels of early (AN-V+/PI−) and late (AN-V+/PI+) apoptotic, and three inflammatory (CD11b-c+, Ly6G+ and MPO+) cells were lowest in group 1 and significantly progressively reduced in groups 2 to 5 (all p < 0.0001). By days 7, 14, 21, 28, and 60 after CCI, the thresholds of thermal paw withdrawal latency (PWL) and mechanical paw withdrawal threshold (PWT) were highest in group 1 and significantly progressively increased in groups 2 to 5 (all p < 0.0001). Numbers of pain-connived cells (Nav1.8+/peripherin+, p-ERK+/peripherin+, p-p38+/peripherin+ and p-p38+/NF200+) and protein expressions of inflammatory (p-NF-κB, IL-1ß, TNF-α and MMP-9), apoptotic (cleaved-caspase-3, cleaved-PARP), oxidative-stress (NOX-1, NOX-2), DNA-damaged (γ-H2AX) and MAPK-family (p-P38, p-JNK, p-ERK1/2) biomarkers as well as the protein levels of Nav.1.3, Nav.1.8, and Nav.1.9 in L4-L5 in dorsal root ganglia displayed an opposite pattern of mechanical PWT among the groups (all p < 0.0001). In conclusion, combined probiotic and ADMSC therapy was superior to merely one for alleviating CCI-induced NP mainly through suppressing inflammation and oxidative stress.

PRPH2 mutation update: In silico assessment of 245 reported and 7 novel variants in patients with retinal disease.

Mutations in PRPH2, encoding peripherin-2, are associated with the development of a wide variety of inherited retinal diseases (IRDs). To determine the causality of the many PRPH2 variants that have been discovered over the last decades, we surveyed all published PRPH2 variants up to July 2020, describing 720 index patients that in total carried 245 unique variants. In addition, we identified seven novel PRPH2 variants in eight additional index patients. The pathogenicity of all variants was determined using the ACMG guidelines. With this, 107 variants were classified as pathogenic, 92 as likely pathogenic, one as benign, and two as likely benign. The remaining 50 variants were classified as variants of uncertain significance. Interestingly, of the total 252 PRPH2 variants, more than half (n = 137) were missense variants. All variants were uploaded into the Leiden Open source Variation and ClinVar databases. Our study underscores the need for experimental assays for variants of unknown significance to improve pathogenicity classification, which would allow us to better understand genotype-phenotype correlations, and in the long-term, hopefully also support the development of therapeutic strategies for patients with PRPH2-associated IRD.

Prph2 initiates outer segment morphogenesis but maturation requires Prph2/Rom1 oligomerization.

The retinal disease gene peripherin 2 (PRPH2) is essential for the formation of photoreceptor outer segments (OSs), where it functions in oligomers with and without its homologue ROM1. However, the precise role of these proteins in OS morphogenesis is not understood. By utilizing a knock-in mouse expressing a chimeric protein comprised of the body of Rom1 and the C-terminus of Prph2 (termed RRCT), we find that the Prph2 C-terminus is necessary and sufficient for the initiation of OSs, while OS maturation requires the body of Prph2 and associated large oligomers. Importantly, dominant-negative physiological and biochemical defects in RRCT heterozygous rods are rescued by removing Rom1, suggesting Rom1 is a regulator for OS formation. Our experiments evaluating Prph2 trafficking show that Rom1 is a key determinant of whether Prph2 complexes utilize conventional versus unconventional (Golgi bypass) secretory pathways to reach the OS. These findings significantly advance our understanding of the molecular underpinnings of OS morphogenesis and particularly the role of Rom1.

Multimeric conformation of type III intermediate filaments but not the filamentous conformation exhibits high affinity to lipid bilayers.

Vimentin, desmin, glial fibrillary acidic protein (GFAP) and peripherin, classified as the type III intermediate filament family, maintain the integrity and architecture of various cell types. Recently, we reported their cell surface expression and binding to multivalent N-acetylglucosamine-conjugated polymers. Furthermore, the presence of vimentin on the surface of various cell types including malignant tumor cells and fibroblasts has been demonstrated. Type III intermediate filament proteins are traditionally considered intracellular proteins and do not possess signal peptides for cell membrane recruitment. Therefore, the mechanism of their transport to the cell surface is unclear. In the current study, we aimed to elucidate this mechanism by focusing on the relationship between their multimeric structure and lipid bilayer affinity. Blue native polyacrylamide gel electrophoresis demonstrated that cell surface-expressed type III intermediate filament proteins formed a multimeric mostly including 4-12-mers but not filamentous structure. Moreover, surface plasmon resonance analysis revealed that the multimeric structure of these recombinant proteins had high affinity to lipid bilayers, whereas their filament-like large multimeric structure did not. Our results suggest that type III intermediate filaments are incorporated into the cell membrane through alteration from a filamentous to a multimeric structure.

Novel molecular mechanisms for Prph2-associated pattern dystrophy.

Mutations in peripherin 2 (PRPH2) have been associated with retinitis pigmentosa (RP) and macular/pattern dystrophies, but the origin of this phenotypic variability is unclear. The majority of Prph2 mutations are located in the large intradiscal loop (D2), a region that contains seven cysteines involved in intra- and intermolecular disulfide bonding and protein folding. A mutation at cysteine 213, which is engaged in an intramolecular disulfide bond, leads to butterfly-shaped pattern dystrophy in humans, in sharp contrast to mutations in the adjacent cysteine at position 214 which result in RP. To help understand this unexpected phenotypic variability, we generated a knockin mouse line carrying the C213Y disease mutation. The mutant Prph2 protein lost the ability to oligomerize with rod outer segment membrane protein 1 (Rom1), but retained the ability to form homotetramers. C213Y heterozygotes had significantly decreased overall Prph2 levels as well as decreased rod and cone function. Critically, supplementation with extra wild-type Prph2 protein elicited improvements in Prph2 protein levels and rod outer segment structure, but not functional rescue in rods or cones. These findings suggest that not all interruptions of D2 loop intramolecular disulfide bonding lead to haploinsufficiency-related RP, but rather that more subtle changes can lead to mutant proteins stable enough to exert gain-of-function defects in rods and cones. This outcome highlights the difficulty in targeting Prph2-associated gain-of-function disease and suggests that elimination of the mutant protein will be a pre-requisite for any curative therapeutic strategy.