A blood-brain barrier-penetrant AAV gene therapy improves neurological function in symptomatic mucolipidosis IV mice.

Mucolipidosis IV (MLIV) is a rare, autosomal recessive, lysosomal disease characterized by intellectual disability, motor deficits, and progressive vision loss. Using adeno-associated vector 9 (AAV9) and AAV-PHP.B as delivery vectors, we previously demonstrated the feasibility of modifying disease course in a mouse model of MLIV by the human MCOLN1 gene transfer. Here, using a primate-enabling capsid AAV.CPP.16 (CPP16), we constructed a new, clinic-oriented MCOLN1 gene expression vector and demonstrated its efficacy in the preclinical model of MLIV. Systemic administration of CPP16-MCOLN1 in adult symptomatic Mcoln1 -/- mice at a dose of 1e12 vg per mouse resulted in MCOLN1 expression in the brain and peripheral tissues, alleviated brain pathology, rescued neuromotor function, and completely prevented paralysis. Notable expression of MCOLN1 transcripts was also detected in the retina of the mouse, which had exhibited significant degeneration at the time of the treatment. However, no increase in retinal thickness was observed after gene therapy treatment. Our results suggest a new AAV-based systemic gene replacement therapy for the treatment of MLIV that could be translated into clinical studies.

Transcriptome analysis in a humanized mouse model of familial dysautonomia reveals tissue-specific gene expression disruption in the peripheral nervous system.

Familial dysautonomia (FD) is a rare recessive neurodevelopmental disease caused by a splice mutation in the Elongator acetyltransferase complex subunit 1 (ELP1) gene. This mutation results in a tissue-specific reduction of ELP1 protein, with the lowest levels in the central and peripheral nervous systems (CNS and PNS, respectively). FD patients exhibit complex neurological phenotypes due to the loss of sensory and autonomic neurons. Disease symptoms include decreased pain and temperature perception, impaired or absent myotatic reflexes, proprioceptive ataxia, and progressive retinal degeneration. While the involvement of the PNS in FD pathogenesis has been clearly recognized, the underlying mechanisms responsible for the preferential neuronal loss remain unknown. In this study, we aimed to elucidate the molecular mechanisms underlying FD by conducting a comprehensive transcriptome analysis of neuronal tissues from the phenotypic mouse model TgFD9; Elp1Δ20/flox. This mouse recapitulates the same tissue-specific ELP1 mis-splicing observed in patients while modeling many of the disease manifestations. Comparison of FD and control transcriptomes from dorsal root ganglion (DRG), trigeminal ganglion (TG), medulla (MED), cortex, and spinal cord (SC) showed significantly more differentially expressed genes (DEGs) in the PNS than the CNS. We then identified genes that were tightly co-expressed and functionally dependent on the level of full-length ELP1 transcript. These genes, defined as ELP1 dose-responsive genes, were combined with the DEGs to generate tissue-specific dysregulated FD signature genes and networks. Within the PNS networks, we observed direct connections between Elp1 and genes involved in tRNA synthesis and genes related to amine metabolism and synaptic signaling. Importantly, transcriptomic dysregulation in PNS tissues exhibited enrichment for neuronal subtype markers associated with peptidergic nociceptors and myelinated sensory neurons, which are known to be affected in FD. In summary, this study has identified critical tissue-specific gene networks underlying the etiology of FD and provides new insights into the molecular basis of the disease.

Transcriptome analysis in a humanized mouse model of familial dysautonomia reveals tissue-specific gene expression disruption in the peripheral nervous system.

Familial dysautonomia (FD) is a rare recessive neurodevelopmental disease caused by a splice mutation in the Elongator acetyltransferase complex subunit 1 ( ELP1 ) gene. This mutation results in a tissue-specific reduction of ELP1 protein, with the lowest levels in the central and peripheral nervous systems (CNS and PNS, respectively). FD patients exhibit complex neurological phenotypes due to the loss of sensory and autonomic neurons. Disease symptoms include decreased pain and temperature perception, impaired or absent myotatic reflexes, proprioceptive ataxia, and progressive retinal degeneration. While the involvement of the PNS in FD pathogenesis has been clearly recognized, the underlying mechanisms responsible for the preferential neuronal loss remain unknown. In this study, we aimed to elucidate the molecular mechanisms underlying FD by conducting a comprehensive transcriptome analysis of neuronal tissues from the phenotypic mouse model TgFD9 ; Elp1 Δ 20/flox . This mouse recapitulates the same tissue-specific ELP1 mis-splicing observed in patients while modeling many of the disease manifestations. Comparison of FD and control transcriptomes from dorsal root ganglion (DRG), trigeminal ganglion (TG), medulla (MED), cortex, and spinal cord (SC) showed significantly more differentially expressed genes (DEGs) in the PNS than the CNS. We then identified genes that were tightly co-expressed and functionally dependent on the level of full-length ELP1 transcript. These genes, defined as ELP1 dose-responsive genes, were combined with the DEGs to generate tissue-specific dysregulated FD signature genes and networks. Within the PNS networks, we observed direct connections between Elp1 and genes involved in tRNA synthesis and genes related to amine metabolism and synaptic signaling. Importantly, transcriptomic dysregulation in PNS tissues exhibited enrichment for neuronal subtype markers associated with peptidergic nociceptors and myelinated sensory neurons, which are known to be affected in FD. In summary, this study has identified critical tissue-specific gene networks underlying the etiology of FD and provides new insights into the molecular basis of the disease.

A Novel Mouse Model for Late-Onset Retinal Degeneration (L-ORD) Develops RPE Abnormalities Due to the Loss of C1qtnf5/Ctrp5.

Late-onset retinal degeneration (L-ORD) is an autosomal dominant macular dystrophy resulting from mutations in the gene CTRP5/C1QTNF5. A mouse model (Ctrp5+/-) for the most common S163R developed many features of human clinical disease. We generated a novel homozygous Ctrp5 gene knock-out (Ctrp5-/-) mouse model to further study the mechanism of L-ORD. The retinal morphology of these mice was evaluated by retinal imaging, light microscopy, and transmission electron microscopy (TEM) at 6, 11, and 18.5 mo. Expression of Ctrp5 was analyzed using immunostaining and qRT-PCR. The Ctrp5-/- mice showed lack of both Ctrp5 transcript and protein. Presence of a significantly larger number of autofluorescent spots was observed in Ctrp5-/- mice compared to the WT (P < 0.0001) at 19 mo. Increased RPE stress with vacuolization and thinning was observed as early as 6 mo in Ctrp5-/- mice. Further, ultrastructural analyses revealed a progressive accumulation of basal laminar sub-RPE deposits in Ctrp5-/- mice from 11 mo. The Ctrp5-/- mice shared retinal and RPE pathology that matches with that previously described for Ctrp5+/- mice suggesting that pathology in these mice results from the loss of functional CTRP5 and that the presence of CTRP5 is critical for normal RPE and retinal function.

Development of an oral treatment that rescues gait ataxia and retinal degeneration in a phenotypic mouse model of familial dysautonomia.

Familial dysautonomia (FD) is a rare neurodegenerative disease caused by a splicing mutation in elongator acetyltransferase complex subunit 1 (ELP1). This mutation leads to the skipping of exon 20 and a tissue-specific reduction of ELP1, mainly in the central and peripheral nervous systems. FD is a complex neurological disorder accompanied by severe gait ataxia and retinal degeneration. There is currently no effective treatment to restore ELP1 production in individuals with FD, and the disease is ultimately fatal. After identifying kinetin as a small molecule able to correct the ELP1 splicing defect, we worked on its optimization to generate novel splicing modulator compounds (SMCs) that can be used in individuals with FD. Here, we optimize the potency, efficacy, and bio-distribution of second-generation kinetin derivatives to develop an oral treatment for FD that can efficiently pass the blood-brain barrier and correct the ELP1 splicing defect in the nervous system. We demonstrate that the novel compound PTC258 efficiently restores correct ELP1 splicing in mouse tissues, including brain, and most importantly, prevents the progressive neuronal degeneration that is characteristic of FD. Postnatal oral administration of PTC258 to the phenotypic mouse model TgFD9;Elp1Δ20/flox increases full-length ELP1 transcript in a dose-dependent manner and leads to a 2-fold increase in functional ELP1 in the brain. Remarkably, PTC258 treatment improves survival, gait ataxia, and retinal degeneration in the phenotypic FD mice. Our findings highlight the great therapeutic potential of this novel class of small molecules as an oral treatment for FD.

Selective retinal ganglion cell loss and optic neuropathy in a humanized mouse model of familial dysautonomia.

Familial dysautonomia (FD) is an autosomal recessive neurodegenerative disease caused by a splicing mutation in the gene encoding Elongator complex protein 1 (ELP1, also known as IKBKAP). This mutation results in tissue-specific skipping of exon 20 with a corresponding reduction of ELP1 protein, predominantly in the central and peripheral nervous system. Although FD patients have a complex neurological phenotype caused by continuous depletion of sensory and autonomic neurons, progressive visual decline leading to blindness is one of the most problematic aspects of the disease, as it severely affects their quality of life. To better understand the disease mechanism as well as to test the in vivo efficacy of targeted therapies for FD, we have recently generated a novel phenotypic mouse model, TgFD9; IkbkapΔ20/flox. This mouse exhibits most of the clinical features of the disease and accurately recapitulates the tissue-specific splicing defect observed in FD patients. Driven by the dire need to develop therapies targeting retinal degeneration in FD, herein, we comprehensively characterized the progression of the retinal phenotype in this mouse, and we demonstrated that it is possible to correct ELP1 splicing defect in the retina using the splicing modulator compound (SMC) BPN-15477.

Developmental regulation of neuronal gene expression by Elongator complex protein 1 dosage.

Familial dysautonomia (FD), a hereditary sensory and autonomic neuropathy, is caused by a mutation in the Elongator complex protein 1 (ELP1) gene that leads to a tissue-specific reduction of ELP1 protein. Our work to generate a phenotypic mouse model for FD headed to the discovery that homozygous deletion of the mouse Elp1 gene leads to embryonic lethality prior to mid-gestation. Given that FD is caused by a reduction, not loss, of ELP1, we generated two new mouse models by introducing different copy numbers of the human FD ELP1 transgene into the Elp1 knockout mouse (Elp1-/-) and observed that human ELP1 expression rescues embryonic development in a dose-dependent manner. We then conducted a comprehensive transcriptome analysis in mouse embryos to identify genes and pathways whose expression correlates with the amount of ELP1. We found that ELP1 is essential for the expression of genes responsible for nervous system development. Further, gene length analysis of the differentially expressed genes showed that the loss of Elp1 mainly impacts the expression of long genes and that by gradually restoring Elongator, their expression is progressively rescued. Finally, through evaluation of co-expression modules, we identified gene sets with unique expression patterns that depended on ELP1 expression.

A deep learning approach to identify gene targets of a therapeutic for human splicing disorders.

Pre-mRNA splicing is a key controller of human gene expression. Disturbances in splicing due to mutation lead to dysregulated protein expression and contribute to a substantial fraction of human disease. Several classes of splicing modulator compounds (SMCs) have been recently identified and establish that pre-mRNA splicing represents a target for therapy. We describe herein the identification of BPN-15477, a SMC that restores correct splicing of ELP1 exon 20. Using transcriptome sequencing from treated fibroblast cells and a machine learning approach, we identify BPN-15477 responsive sequence signatures. We then leverage this model to discover 155 human disease genes harboring ClinVar mutations predicted to alter pre-mRNA splicing as targets for BPN-15477. Splicing assays confirm successful correction of splicing defects caused by mutations in CFTR, LIPA, MLH1 and MAPT. Subsequent validations in two disease-relevant cellular models demonstrate that BPN-15477 increases functional protein, confirming the clinical potential of our predictions.

Late-onset retinal degeneration pathology due to mutations in CTRP5 is mediated through HTRA1.

Late-onset retinal degeneration (L-ORD) is an autosomal dominant macular degeneration characterized by the formation of sub-retinal pigment epithelium (RPE) deposits and neuroretinal atrophy. L-ORD results from mutations in the C1q-tumor necrosis factor-5 protein (CTRP5), encoded by the CTRP5/C1QTNF5 gene. To understand the mechanism underlying L-ORD pathology, we used a human cDNA library yeast two-hybrid screen to identify interacting partners of CTRP5. Additionally, we analyzed the Bruch's membrane/choroid (BM-Ch) from wild-type (Wt), heterozygous S163R Ctrp5 mutation knock-in (Ctrp5S163R/wt ), and homozygous knock-in (Ctrp5S163R/S163R ) mice using mass spectrometry. Both approaches showed an association between CTRP5 and HTRA1 via its C-terminal PDZ-binding motif, stimulation of the HTRA1 protease activity by CTRP5, and CTRP5 serving as an HTRA1 substrate. The S163R-CTRP5 protein also binds to HTRA1 but is resistant to HTRA1-mediated cleavage. Immunohistochemistry and proteomic analysis showed significant accumulation of CTRP5 and HTRA1 in BM-Ch of Ctrp5S163R/S163R and Ctrp5S163R/wt mice compared with Wt. Additional extracellular matrix (ECM) components that are HTRA1 substrates also accumulated in these mice. These results implicate HTRA1 and its interaction with CTRP5 in L-ORD pathology.

Long-Term Effects of Gene Therapy in a Novel Mouse Model of Human MFRP-Associated Retinopathy.

Patients harboring homozygous c.498_499insC mutations in MFRP demonstrate hyperopia, microphthalmia, retinitis pigmentosa, retinal pigment epithelial atrophy, variable degrees of foveal edema, and optic disc drusen. The disease phenotype is variable, however, with some patients maintaining good central vision and cone function till late in the disease. A knock-in mouse model with the c.498_499insC mutation in Mfrp (Mfrp KI/KI) was developed to understand the effects of these mutations in the retina. The model shares many of the features of human clinical disease, including reduced axial length, hyperopia, retinal degeneration, retinal pigment epithelial atrophy, and decreased electrophysiological responses. In addition, the eyes of these mice had a significantly greater refractive error (p < 0.01) when compared to age-matched wild-type control animals. Administration of recombinant adeno-associated virus-mediated Mfrp gene therapy significantly prevented thinning from retinal neurodegeneration (p < 0.005) and preserved retinal electrophysiology (p < 0.001) when treated eyes were compared to contralateral sham-treated control eyes. The Mfrp KI/KI mice will serve as a useful tool to model human disease and point to a potential gene therapeutic approach for patients with preserved vision and electrophysiological responses in MFRP-related retinopathy.

IFT88 mutations identified in individuals with non-syndromic recessive retinal degeneration result in abnormal ciliogenesis.

Whole genome sequencing (WGS) was performed to identify the variants responsible for inherited retinal degeneration (IRD) in a Caucasian family. Segregation analysis of selected rare variants with pathogenic potential identified a set of compound heterozygous changes p.Arg266*:c.796C>T and p.Ala568Thr:c.1702G>A in the intraflagellar transport protein-88 (IFT88) gene segregating with IRD. Expression of IFT88 with the p.Arg266* and p.Ala568Thr mutations in mIMDC3 cells by transient transfection and in HeLa cells by introducing the mutations using CRISPR-cas9 system suggested that both mutations result in the formation of abnormal ciliary structures. The introduction of the IFT88 p.Arg266* variant in the homozygous state in HeLa cells by CRISPR-Cas9 genome-editing revealed that the mutant transcript undergoes nonsense-mediated decay leading to a significant depletion of IFT88 transcript. Additionally, abnormal ciliogenesis was observed in these cells. These observations suggest that the rare and unique combination of IFT88 alleles observed in this study provide insight into the physiological role of IFT88 in humans and the likely mechanism underlying retinal pathology in the pedigree with IRD.

Glucotoxicity promotes aberrant activation and mislocalization of Ras-related C3 botulinum toxin substrate 1 [Rac1] and metabolic dysfunction in pancreatic islet ß-cells: reversal of such metabolic defects by metformin.

Emerging evidence suggests that long-term exposure of insulin-secreting pancreatic ß-cells to hyperglycemic (HG; glucotoxic) conditions promotes oxidative stress, which, in turn, leads to stress kinase activation, mitochondrial dysfunction, loss of nuclear structure and integrity and cell apoptosis. Original observations from our laboratory have proposed that Rac1 plays a key regulatory role in the generation of oxidative stress and downstream signaling events culminating in the onset of dysfunction of pancreatic ß-cells under the duress of metabolic stress. However, precise molecular and cellular mechanisms underlying the metabolic roles of hyperactive Rac1 remain less understood. Using pharmacological and molecular biological approaches, we now report mistargetting of biologically-active Rac1 [GTP-bound conformation] to the nuclear compartment in clonal INS-1 cells, normal rat islets and human islets under HG conditions. Our findings also suggest that such a signaling step is independent of post-translational prenylation of Rac1. Evidence is also presented to highlight novel roles for sustained activation of Rac1 in HG-induced expression of Cluster of Differentiation 36 [CD36], a fatty acid transporter protein, which is implicated in cell apoptosis. Finally, our findings suggest that metformin, a biguanide anti-diabetic drug, at a clinically relevant concentration, prevents ß-cell defects [Rac1 activation, nuclear association, CD36 expression, stress kinase and caspase-3 activation, and loss in metabolic viability] under the duress of glucotoxicity. Potential implications of these findings in the context of novel and direct regulation of islet ß-cell function by metformin are discussed.

TopoisomeraseIIß in HIV-1 transactivation.

TopoisomeraseIIß, an isoform of type II topoisomerase, was found to be functional in various viral infections. Its plausible role in HIV life cycle was also suggested earlier, but not clearly established. In the present study, we have investigated the role of TopoIIß in HIV-1 infection by its gain and loss of function. Overexpression of TopoIIß lead to an increase in viral replication, resulting in enhanced virion production. HIV-1 replication was impaired when TopoIIß was down regulated by siRNA and inhibited by ICRF-193 and merbarone. The role of TopoIIß in HIV-1 transcription was shown through its interaction with Tat and recruitement to long terminal repeat (LTR) region by co-immunoprecipitation and ChIP assays. Involvement of TopoIIß in transactivation of HIV-1 LTR was confirmed by luciferase assay in reporter cell line, TZM bl and also by transfection of reporter exogenously. It was also observed that LTR transactivation commensurated with the expression of TopoIIß in the presence of Tat. In addition, a decreased viral gene expression on treatment with merbarone exemplifies the importance of catalytic activity of TopoIIß in viral replication. These observations indicate that TopoIIß is involved in the cascade of coactivator complexes that are recruited to LTR for regulation of HIV-1 transcription.

Analysis of gene expression during aging of CGNs in culture: implication of SLIT2 and NPY in senescence.

Senescence is the major key factor that leads to the loss of neurons throughout aging. Cellular senescence is not the consequence of single cause, but there are multiple aspects which may induce senescence in a cell. Various causes such as gene expression, molecular interactions and protein processing and chromatin organization are described as causal factor for senescence. It is well known that the damage to the nuclear or mitochondrial DNA contributes to the aging either directly by inducing the apoptosis/cellular senescence or indirectly by altering cellular functions. The significant nuclear DNA damage with the age is directly associated with the continuous declining in DNA repair. The continuous decline in expression of topoisomerase 2 beta (Topo IIß) in cultured cerebellar granule neurons over time indicated the decline in the repair of damage DNA. DNA Topo IIß is an enzyme that is crucial for solving topological problems of DNA and thus has an important role in DNA repair. The enzyme is predominantly present in non-proliferating cells such as neurons. In this paper, we have studied the genes which were differentially expressed over time in cultured cerebellar granule neurons (CGNs) and identified potential genes associated with the senescence. Our results showed that the two genes neuropeptide Y (Npy) and Slit homolog 2 (Drosophila) (Slit2) gradually increase during aging, and upon suppression of these two genes, there was gradual increase in cell viability along with restoration of the expression of Topo IIß and potential repair proteins.

Evolutionary and molecular facts link the WWC protein family to Hippo signaling.

The scaffolding protein KIBRA (also called WWC1) is involved in the regulation of important intracellular transport processes and the establishment of cell polarity. Furthermore, KIBRA/WWC1 is an upstream regulator of the Hippo signaling pathway that controls cell proliferation and organ size in animals. KIBRA/WWC1 represents only one member of the WWC protein family that also includes the highly similar proteins WWC2 and WWC3. Although the function of KIBRA/WWC1 was studied intensively in cells and animal models, the importance of WWC2 and WWC3 was not yet elucidated. Here, we describe evolutionary, molecular, and functional aspects of the WWC family. We show that the WWC genes arose in the ancestor of bilateral animals (clades such as insects and vertebrates) from a single founder gene most similar to the present KIBRA/WWC1-like sequence of Drosophila. This situation was still maintained until the common ancestor of lancelet and vertebrates. In fish, a progenitor-like sequence of mammalian KIBRA/WWC1 and WWC2 is expressed together with WWC3. Finally, in all tetrapods, the three family members, KIBRA/WWC1, WWC2, and WWC3, are found, except for a large genomic deletion including WWC3 in Mus musculus. At the molecular level, the highly conserved WWC proteins share a similar primary structure, the ability to form homo- and heterodimers and the interaction with a common set of binding proteins. Furthermore, all WWC proteins negatively regulate cell proliferation and organ growth due to a suppression of the transcriptional activity of YAP, the major effector of the Hippo pathway.

Topoisomerase IIß associates with Ku70 and PARP-1 during double strand break repair of DNA in neurons.

In the present study, the activity of Topoisomerase IIß (TopoIIß) is evaluated during peroxide induced double stranded DNA breaks (DSBs) repair in primary neurons. The results showed that the TopoIIß levels were enhanced during recovery from peroxide mediated damage (PED) along with Ku70, PARP-1, pol beta, and WRN helicase. Furthermore, siRNA mediated knock-down of TopoIIß in primary neurons conferred enhanced susceptibility to PED in neurons. DSBs in neurons are repaired through two pathways, one promoted by Ku70, while the other is by PARP-1 dependent manner. Participation of TopoIIß in both pathways was assessed by analysis of the interaction of TopoIIß with Ku70 and PARP-1 using co-immunoprecipitation experiments in extracts of neurons under peroxide treatment and recovery. The results of these studies showed a strong interaction of TopoIIß with Ku70 as well as PARP-1 suggesting that TopoIIß is associated both in Ku70 and PARP-dependent pathways in DSBs repair in primary neurons. The study has thus established that TopoIIß is an essential component in DSBs repair in primary neurons in both Ku70 and PARP-1 dependent pathways. We suppose that the interaction of TopoIIß may provide stabilization of the repair complex, which may assist in maintenance of tensional integrity in genomic DNA.