AAV-mediated ERdj5 overexpression protects against P23H rhodopsin toxicity.

Rhodopsin misfolding caused by the P23H mutation is a major cause of autosomal dominant retinitis pigmentosa (adRP). To date, there are no effective treatments for adRP. The BiP co-chaperone and reductase ERdj5 (DNAJC10) is part of the endoplasmic reticulum (ER) quality control machinery, and previous studies have shown that overexpression of ERdj5 in vitro enhanced the degradation of P23H rhodopsin, whereas knockdown of ERdj5 increased P23H rhodopsin ER retention and aggregation. Here, we investigated the role of ERdj5 in photoreceptor homeostasis in vivo by using an Erdj5 knockout mouse crossed with the P23H knock-in mouse and by adeno-associated viral (AAV) vector-mediated gene augmentation of ERdj5 in P23H-3 rats. Electroretinogram (ERG) and optical coherence tomography of Erdj5-/- and P23H+/-:Erdj5-/- mice showed no effect of ERdj5 ablation on retinal function or photoreceptor survival. Rhodopsin levels and localization were similar to those of control animals at a range of time points. By contrast, when AAV2/8-ERdj5-HA was subretinally injected into P23H-3 rats, analysis of the full-field ERG suggested that overexpression of ERdj5 reduced visual function loss 10 weeks post-injection (PI). This correlated with a significant preservation of photoreceptor cells at 4 and 10 weeks PI. Assessment of the outer nuclear layer (ONL) morphology showed preserved ONL thickness and reduced rhodopsin retention in the ONL in the injected superior retina. Overall, these data suggest that manipulation of the ER quality control and ER-associated degradation factors to promote mutant protein degradation could be beneficial for the treatment of adRP caused by mutant rhodopsin.

Arl3 and RP2 regulate the trafficking of ciliary tip kinesins.

Ciliary trafficking defects are the underlying cause of many ciliopathies, including Retinitis Pigmentosa (RP). Anterograde intraflagellar transport (IFT) is mediated by kinesin motor proteins; however, the function of the homodimeric Kif17 motor in cilia is poorly understood, whereas Kif7 is known to play an important role in stabilizing cilia tips. Here we identified the ciliary tip kinesins Kif7 and Kif17 as novel interaction partners of the small GTPase Arl3 and its regulatory GTPase activating protein (GAP) Retinitis Pigmentosa 2 (RP2). We show that Arl3 and RP2 mediate the localization of GFP-Kif17 to the cilia tip and competitive binding of RP2 and Arl3 with Kif17 complexes. RP2 and Arl3 also interact with another ciliary tip kinesin, Kif7, which is a conserved regulator of Hedgehog (Hh) signaling. siRNA-mediated loss of RP2 or Arl3 reduced the level of Kif7 at the cilia tip. This was further validated by reduced levels of Kif7 at cilia tips detected in fibroblasts and induced pluripotent stem cell (iPSC) 3D optic cups derived from a patient carrying an RP2 nonsense mutation c.519C > T (p.R120X), which lack detectable RP2 protein. Translational read-through inducing drugs (TRIDs), such as PTC124, were able to restore Kif7 levels at the ciliary tip of RP2 null cells. Collectively, our findings suggest that RP2 and Arl3 regulate the trafficking of specific kinesins to cilia tips and provide additional evidence that TRIDs could be clinically beneficial for patients with this retinal degeneration.

Protein kinase CK2 modulates HSJ1 function through phosphorylation of the UIM2 domain.

HSJ1 (DNAJB2), a member of the DNAJ family of molecular chaperones, is a key player in neuronal proteostasis maintenance. It binds ubiquitylated proteins through its Ubiquitin Interacting Motifs (UIMs) and facilitates their delivery to the proteasome for degradation. Mutations in the DNAJB2 gene lead to inherited neuropathies such as Charcot-Marie-Tooth type-2, distal hereditary motor neuropathies, spinal muscular atrophy with parkinsonism and the later stages can resemble amyotrophic lateral sclerosis. HSJ1 overexpression can reduce aggregation of neurodegeneration-associated proteins in vitro and in vivo; however, the regulation of HSJ1 function is little understood. Here we show that CK2, a ubiquitous and constitutively active protein kinase, phosphorylates HSJ1 within its second UIM, at the dominant site Ser250 and the hierarchical site Ser247. A phospho-HSJ1 specific antibody confirmed phosphorylation of endogenous HSJ1a and HSJ1b. A tandem approach of phospho-site mutation and treatment with CK2 specific inhibitors demonstrated that phosphorylation at these sites is accompanied by a reduced ability of HSJ1 to bind ubiquitylated clients and to exert its chaperone activity. Our results disclose a novel interplay between ubiquitin- and phosphorylation-dependent signalling, and represent the first report of a regulatory mechanism for UIM-dependent function. They also suggest that CK2 inhibitors could release the full neuroprotective potential of HSJ1, and deserve future interest as therapeutic strategies for neurodegenerative disease.

REEP6 deficiency leads to retinal degeneration through disruption of ER homeostasis and protein trafficking.

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy. We recently identified mutations in REEP6, which encodes the receptor expression enhancing protein 6, in several families with autosomal recessive RP. REEP6 is related to the REEP and Yop1p family of ER shaping proteins and potential receptor accessory proteins, but the role of REEP6 in the retina is unknown. Here we characterize the disease mechanisms associated with loss of REEP6 function using a Reep6 knockout mouse generated by CRISPR/Cas9 gene editing. In control mice REEP6 was localized to the inner segment and outer plexiform layer of rod photoreceptors. The Reep6-/- mice exhibited progressive photoreceptor degeneration from P20 onwards. Ultrastructural analyses at P20 by transmission electron microscopy and 3View serial block face scanning EM revealed an expansion of the distal ER in the Reep6-/- rods and an increase in their number of mitochondria. Electroretinograms revealed photoreceptor dysfunction preceded degeneration, suggesting potential defects in phototransduction. There was no effect on the traffic of rhodopsin, Rom1 or peripherin/rds; however, the retinal guanylate cyclases GC1 and GC2 were severely affected in the Reep6 knockout animals, with almost undetectable expression. These changes correlated with an increase in C/EBP homologous protein (CHOP) expression and the activation of caspase 12, suggesting that ER stress contributes to cell death. Collectively, these data suggest that REEP6 plays an essential role in maintaining cGMP homeostasis though facilitating the stability and/or trafficking of guanylate cyclases and maintaining ER and mitochondrial homeostasis.

Mutations in REEP6 Cause Autosomal-Recessive Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is the most frequent form of inherited retinal dystrophy. RP is genetically heterogeneous and the genes identified to date encode proteins involved in a wide range of functional pathways, including photoreceptor development, phototransduction, the retinoid cycle, cilia, and outer segment development. Here we report the identification of biallelic mutations in Receptor Expression Enhancer Protein 6 (REEP6) in seven individuals with autosomal-recessive RP from five unrelated families. REEP6 is a member of the REEP/Yop1 family of proteins that influence the structure of the endoplasmic reticulum but is relatively unstudied. The six variants identified include three frameshift variants, two missense variants, and a genomic rearrangement that disrupts exon 1. Human 3D organoid optic cups were used to investigate REEP6 expression and confirmed the expression of a retina-specific isoform REEP6.1, which is specifically affected by one of the frameshift mutations. Expression of the two missense variants (c.383C>T [p.Pro128Leu] and c.404T>C [p.Leu135Pro]) and the REEP6.1 frameshift mutant in cultured cells suggest that these changes destabilize the protein. Furthermore, CRISPR-Cas9-mediated gene editing was used to produce Reep6 knock-in mice with the p.Leu135Pro RP-associated variant identified in one RP-affected individual. The homozygous knock-in mice mimic the clinical phenotypes of RP, including progressive photoreceptor degeneration and dysfunction of the rod photoreceptors. Therefore, our study implicates REEP6 in retinal homeostasis and highlights a pathway previously uncharacterized in retinal dystrophy.

Using induced pluripotent stem cells to understand retinal ciliopathy disease mechanisms and develop therapies.

The photoreceptor cells in the retina have a highly specialised sensory cilium, the outer segment (OS), which is important for detecting light. Mutations in cilia-related genes often result in retinal degeneration. The ability to reprogramme human cells into induced pluripotent stem cells and then differentiate them into a wide range of different cell types has revolutionised our ability to study human disease. To date, however, the challenge of producing fully differentiated photoreceptors in vitro has limited the application of this technology in studying retinal degeneration. In this review, we will discuss recent advances in stem cell technology and photoreceptor differentiation. In particular, the development of photoreceptors with rudimentary OS that can be used to understand disease mechanisms and as an important model to test potential new therapies for inherited retinal ciliopathies.

Targeting the Proteostasis Network in Rhodopsin Retinitis Pigmentosa.

Mutations in rhodopsin are one of the most common causes of retinitis pigmentosa (RP). Misfolding of rhodopsin can result in disruptions in cellular protein homeostasis, or proteostasis. There is currently no available treatment for RP. In this review, we discuss the different approaches currently being investigated for treatment of rhodopsin RP, focusing on the potential of manipulation of the proteostasis network as a therapeutic approach to combat retinal degeneration.

Mitochondrial dysfunction and Purkinje cell loss in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS).

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset neurological disease resulting from mutations in the SACS gene encoding sacsin, a 4,579-aa protein of unknown function. Originally identified as a founder disease in Québec, ARSACS is now recognized worldwide. Prominent features include pyramidal spasticity and cerebellar ataxia, but the underlying pathology and pathophysiological mechanisms are unknown. We have generated an animal model for ARSACS, sacsin knockout mice, that display age-dependent neurodegeneration of cerebellar Purkinje cells. To explore the pathophysiological basis for this observation, we examined the cell biological properties of sacsin. We show that sacsin localizes to mitochondria in non-neuronal cells and primary neurons and that it interacts with dynamin-related protein 1, which participates in mitochondrial fission. Fibroblasts from ARSACS patients show a hyperfused mitochondrial network, consistent with defects in mitochondrial fission. Sacsin knockdown leads to an overly interconnected and functionally impaired mitochondrial network, and mitochondria accumulate in the soma and proximal dendrites of sacsin knockdown neurons. Disruption of mitochondrial transport into dendrites has been shown to lead to abnormal dendritic morphology, and we observe striking alterations in the organization of dendritic fields in the cerebellum of knockout mice that precedes Purkinje cell death. Our data identifies mitochondrial dysfunction/mislocalization as the likely cellular basis for ARSACS and indicates a role for sacsin in regulation of mitochondrial dynamics.

Deep intronic mutation in OFD1, identified by targeted genomic next-generation sequencing, causes a severe form of X-linked retinitis pigmentosa (RP23).

X-linked retinitis pigmentosa (XLRP) is genetically heterogeneous with two causative genes identified, RPGR and RP2. We previously mapped a locus for a severe form of XLRP, RP23, to a 10.71 Mb interval on Xp22.31-22.13 containing 62 genes. Candidate gene screening failed to identify a causative mutation, so we adopted targeted genomic next-generation sequencing of the disease interval to determine the molecular cause of RP23. No coding variants or variants within or near splice sites were identified. In contrast, a variant deep within intron 9 of OFD1 increased the splice site prediction score 4 bp upstream of the variant. Mutations in OFD1 cause the syndromic ciliopathies orofaciodigital syndrome-1, which is male lethal, Simpson-Golabi-Behmel syndrome type 2 and Joubert syndrome. We tested the effect of the IVS9+706A>G variant on OFD1 splicing in vivo. In RP23 patient-derived RNA, we detected an OFD1 transcript with the insertion of a cryptic exon spliced between exons 9 and 10 causing a frameshift, p.N313fs.X330. Correctly spliced OFD1 was also detected in patient-derived RNA, although at reduced levels (39%), hence the mutation is not male lethal. Our data suggest that photoreceptors are uniquely susceptible to reduced expression of OFD1 and that an alternative disease mechanism can cause XLRP. This disease mechanism of reduced expression for a syndromic ciliopathy gene causing isolated retinal degeneration is reminiscent of CEP290 intronic mutations that cause Leber congenital amaurosis, and we speculate that reduced dosage of correctly spliced ciliopathy genes may be a common disease mechanism in retinal degenerations.

Iron accumulation induces oxidative stress, while depressing inflammatory polarization in human iPSC-derived microglia.

Iron accumulation in microglia has been observed in Alzheimer's disease and other neurodegenerative disorders and is thought to contribute to disease progression through various mechanisms, including neuroinflammation. To study this interaction, we treated human induced pluripotent stem cell-derived microglia (iPSC-MG) with iron, in combination with inflammatory stimuli such as interferon gamma (IFN-γ) and amyloid ß. Both IFN-γ and iron treatment increased labile iron levels, but only iron treatment led to a consistent increase of ferritin levels, reflecting long-term iron storage. Therefore, in iPSC-MG, ferritin appeared to be regulated by iron revels rather than inflammation. Further investigation showed that while IFN-γ induced pro-inflammatory activation, iron treatment dampened both classic pro- and anti-inflammatory activation on a transcriptomic level. Notably, iron-loaded microglia showed strong upregulation of cellular stress response pathways, the NRF2 pathway, and other oxidative stress pathways. Functionally, iPSC-MG exhibited altered phagocytosis and impaired mitochondrial metabolism following iron treatment. Collectively, these data suggest that in MG, in contrast to current hypotheses, iron treatment does not result in pro-inflammatory activation, but rather dampens it and induces oxidative stress.

Amyloid beta accumulations and enhanced neuronal differentiation in cerebral organoids of Dutch-type cerebral amyloid angiopathy patients.

Introduction: ADutch-type cerebral amyloid angiopathy (D-CAA) is a hereditary brain disorder caused by a point mutation in the amyloid precursor protein (APP) gene. The mutation is located within the amyloid beta (Aß) domain of APP and leads to Aß peptide accumulation in and around the cerebral vasculature. There lack of disease models to study the cellular and molecular pathological mechanisms of D-CAA together with the absence of a disease phenotype in vitro in overexpression cell models, as well as the limited availability of D-CAA animal models indicates the need for a D-CAA patient-derived model. Methods: We generated cerebral organoids from four D-CAA patients and four controls, cultured them up to 110 days and performed immunofluorescent and targeted gene expression analyses at two time points (D52 and D110). Results: D-CAA cerebral organoids exhibited Aß accumulations, showed enhanced neuronal and astrocytic gene expression and TGFß pathway de-regulation. Conclusions: These results illustrate the potential of cerebral organoids as in vitro disease model of D-CAA that can be used to understand disease mechanisms of D-CAA and can serve as therapeutic intervention platform for various Aß-related disorders.

The ataxia protein sacsin is a functional co-chaperone that protects against polyglutamine-expanded ataxin-1.

An extensive protein-protein interaction network has been identified between proteins implicated in inherited ataxias. The protein sacsin, which is mutated in the early-onset neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay, is a node in this interactome. Here, we have established the neuronal expression of sacsin and functionally characterized domains of the 4579 amino acid protein. Sacsin is most highly expressed in large neurons, particularly within brain motor systems, including cerebellar Purkinje cells. Its subcellular localization in SH-SY5Y neuroblastoma cells was predominantly cytoplasmic with a mitochondrial component. We identified a putative ubiquitin-like (UbL) domain at the N-terminus of sacsin and demonstrated an interaction with the proteasome. Furthermore, sacsin contains a predicted J-domain, the defining feature of DnaJ/Hsp40 proteins. Using a bacterial complementation assay, the sacsin J-domain was demonstrated to be functional. The presence of both UbL and J-domains in sacsin suggests that it may integrate the ubiquitin-proteasome system and Hsp70 function to a specific cellular role. The Hsp70 chaperone machinery is an important component of the cellular response towards aggregation prone mutant proteins that are associated with neurodegenerative diseases. We therefore investigated the effects of siRNA-mediated sacsin knockdown on polyglutamine-expanded ataxin-1. Importantly, SACS siRNA did not affect cell viability with GFP-ataxin-1[30Q], but enhanced the toxicity of GFP-ataxin-1[82Q], suggesting that sacsin is protective against mutant ataxin-1. Thus, sacsin is an ataxia protein and a regulator of the Hsp70 chaperone machinery that is implicated in the processing of other ataxia-linked proteins.

Detailed Phenotyping and Therapeutic Strategies for Intronic ABCA4 Variants in Stargardt Disease.

Stargardt disease is a progressive retinal disorder caused by bi-allelic mutations in the ABCA4 gene that encodes the ATP-binding cassette, subfamily A, member 4 transporter protein. Over the past few years, we and others have identified several pathogenic variants that reside within the introns of ABCA4, including a recurrent variant in intron 36 (c.5196+1137G>A) of which the pathogenicity so far remained controversial. Detailed clinical characterization of this variant confirmed its pathogenic nature, and classified it as an allele of intermediate severity. Moreover, we discovered several additional ABCA4 variants clustering in intron 36. Several of these variants resulted in aberrant splicing of ABCA4, i.e., the inclusion of pseudoexons, while the splicing defects caused by the recurrent c.5196+1137G>A variant strongly increased upon differentiation of patient-derived induced pluripotent stem cells into retina-like cells. Finally, all splicing defects could be rescued by the administration of antisense oligonucleotides that were designed to specifically block the pseudoexon insertion, including rescue in 3D retinal organoids harboring the c.5196+1137G>A variant. Our data illustrate the importance of intronic variants in ABCA4 and expand the therapeutic possibilities for overcoming splicing defects in Stargardt disease.

DNAJ Proteins in neurodegeneration: essential and protective factors.

Maintenance of protein homeostasis is vitally important in post-mitotic cells, particularly neurons. Neurodegenerative diseases such as polyglutamine expansion disorders-like Huntington's disease or spinocerebellar ataxia (SCA), Alzheimer's disease, fronto-temporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and Parkinson's disease-are often characterized by the presence of inclusions of aggregated protein. Neurons contain complex protein networks dedicated to protein quality control and maintaining protein homeostasis, or proteostasis. Molecular chaperones are a class of proteins with prominent roles in maintaining proteostasis, which act to bind and shield hydrophobic regions of nascent or misfolded proteins while allowing correct folding, conformational changes and enabling quality control. There are many different families of molecular chaperones with multiple functions in proteostasis. The DNAJ family of molecular chaperones is the largest chaperone family and is defined by the J-domain, which regulates the function of HSP70 chaperones. DNAJ proteins can also have multiple other protein domains such as ubiquitin-interacting motifs or clathrin-binding domains leading to diverse and specific roles in the cell, including targeting client proteins for degradation via the proteasome, chaperone-mediated autophagy and uncoating clathrin-coated vesicles. DNAJ proteins can also contain ER-signal peptides or mitochondrial leader sequences, targeting them to specific organelles in the cell. In this review, we discuss the multiple roles of DNAJ proteins and in particular focus on the role of DNAJ proteins in protecting against neurodegenerative diseases caused by misfolded proteins. We also discuss the role of DNAJ proteins as direct causes of inherited neurodegeneration via mutations in DNAJ family genes.This article is part of the theme issue 'Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective'.

Splice-Modulating Oligonucleotide QR-110 Restores CEP290 mRNA and Function in Human c.2991+1655A>G LCA10 Models.

Leber congenital amaurosis type 10 (LCA10) is a severe inherited retinal dystrophy associated with mutations in CEP290. The deep intronic c.2991+1655A>G mutation in CEP290 is the most common mutation in LCA10 individuals and represents an ideal target for oligonucleotide therapeutics. Here, a panel of antisense oligonucleotides was designed to correct the splicing defect associated with the mutation and screened for efficacy and safety. This identified QR-110 as the best-performing molecule. QR-110 restored wild-type CEP290 mRNA and protein expression levels in CEP290 c.2991+1655A>G homozygous and compound heterozygous LCA10 primary fibroblasts. Furthermore, in homozygous three-dimensional iPSC-derived retinal organoids, QR-110 showed a dose-dependent restoration of mRNA and protein function, as measured by percentage and length of photoreceptor cilia, without off-target effects. Localization studies in wild-type mice and rabbits showed that QR-110 readily reached all retinal layers, with an estimated half-life of 58 days. It was well tolerated following intravitreal injection in monkeys. In conclusion, the pharmacodynamic, pharmacokinetic, and safety properties make QR-110 a promising candidate for treating LCA10, and clinical development is currently ongoing.

Identification and Correction of Mechanisms Underlying Inherited Blindness in Human iPSC-Derived Optic Cups.

Leber congenital amaurosis (LCA) is an inherited retinal dystrophy that causes childhood blindness. Photoreceptors are especially sensitive to an intronic mutation in the cilia-related gene CEP290, which causes missplicing and premature termination, but the basis of this sensitivity is unclear. Here, we generated differentiated photoreceptors in three-dimensional optic cups and retinal pigment epithelium (RPE) from iPSCs with this common CEP290 mutation to investigate disease mechanisms and evaluate candidate therapies. iPSCs differentiated normally into RPE and optic cups, despite abnormal CEP290 splicing and cilia defects. The highest levels of aberrant splicing and cilia defects were observed in optic cups, explaining the retinal-specific manifestation of this CEP290 mutation. Treating optic cups with an antisense morpholino effectively blocked aberrant splicing and restored expression of full-length CEP290, restoring normal cilia-based protein trafficking. These results provide a mechanistic understanding of the retina-specific phenotypes in CEP290 LCA patients and potential strategies for therapeutic intervention.

The cell stress machinery and retinal degeneration.

Retinal degenerations are a group of clinically and genetically heterogeneous disorders characterised by progressive loss of vision due to neurodegeneration. The retina is a highly specialised tissue with a unique architecture and maintaining homeostasis in all the different retinal cell types is crucial for healthy vision. The retina can be exposed to a variety of environmental insults and stress, including light-induced damage, oxidative stress and inherited mutations that can lead to protein misfolding. Within retinal cells there are different mechanisms to cope with disturbances in proteostasis, such as the heat shock response, the unfolded protein response and autophagy. In this review, we discuss the multiple responses of the retina to different types of stress involved in retinal degenerations, such as retinitis pigmentosa, age-related macular degeneration and glaucoma. Understanding the mechanisms that maintain and re-establish proteostasis in the retina is important for developing new therapeutic approaches to fight blindness.

Deficiency of ferritin heavy-chain nuclear import in triple a syndrome implies nuclear oxidative damage as the primary disease mechanism.

Triple A syndrome is a rare autosomal recessive disorder characterized by ACTH-resistant adrenal failure, alacrima, achalasia, and progressive neurological manifestations. The majority of cases are associated with mutations in the AAAS gene, which encodes a novel, 60-kDa WD-repeat nuclear pore protein, alacrima-achalasia-adrenal insufficiency neurological disorder (ALADIN) of unknown function. Our aim was to elucidate the functional role of ALADIN by determining its interacting protein partners using the bacterial two-hybrid (B2-H) technique. Nonidentical cDNA fragments were identified from both a HeLa S-3 cell and human cerebellar cDNA library that encoded the full-length ferritin heavy chain protein (FTH1). This interaction was confirmed by both co-immunoprecipitation and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer studies. Immunoblotting showed that fibroblasts from triple A patients (with known AAAS mutations) lack nuclear FTH1, suggesting that the nuclear translocation of FTH1 is defective. Cells transfected with FTH1 and visualized by confocal microscopy had very little nuclear FTH1, but when cotransfected with AAAS, FTH1 is readily visible in the nuclei. Therefore, FTH1 nuclear translocation is enhanced when ALADIN is coexpressed in these cells. In addition to its well known iron storage role, FTH1 has been shown to protect the nucleus from oxidative damage. Apoptosis of neuronal cells induced by hydrogen peroxide was significantly reduced by transfection of AAAS or by FTH1 or maximally by both genes together. Taken together, this work offers a plausible mechanism for the progressive clinical features of triple A syndrome.