Therapy for prion diseases: Insights from the use of RNA interference

Insights into the molecular basis and the temporal evolution of neurotoxicity in prion disease are increasing, and recent work in mice leads to new avenues for targeting treatment of these disorders. Using lentivirally mediated RNA interference (RNAi) against native prion protein (PrP), White et al. report the first therapeutic intervention that results in neuronal rescue, prevents symptoms and increases survival in mice with established prion disease.¹ Both the target and the timing of treatment here are crucial to the effectiveness of this strategy: the formation of the neurotoxic prion agent is prevented at a point when diseased neurons can still be saved from death. But the data also give new insights into the timing of treatment in the context of the pattern of spread of prion infection throughout the brain, with implications for developing the most effective treatments.Key words: prion, RNA interference, gene therapy, neurodegeneration, synapticThis perspective considers developments in the field that led to the rationale for targeting endogenous prion protein (PrP) in prion therapeutics and to the discovery of a window of reversibility of early neuronal damage in prion disease. It introduces RNA interference (RNAi) and its therapeutic use in this context and discusses insights into prion pathogenesis and future treatment strategies and goals. A key concept is targeting the critical brain regions for the spread of prion replication. This may have relevance in other neurodegenerative diseases due to protein misfolding, which recent literature suggests may also propagate throughout the brain in disease-specific patterns.     

Natural and experimental prion diseases of humans and animals.

Prions cause transmissible and genetic neurodegenerative diseases. Infectious prion particles are composed largely, if not entirely, of an abnormal isoform of the prion protein (PrPSc), which is encoded by a chromosomal gene. Although the PrP gene is single copy, transgenic mice with both alleles of the PrP gene ablated develop normally. A post-translational process, as yet unidentified, converts the cellular prion protein (PrPC) into PrPSc. Scrapie incubation times, neuropathology and prion synthesis in transgenic mice are controlled by the PrP gene. Mutations in this gene are genetically linked to the development of neurodegeneration. Transgenic mice expressing mutant PrP spontaneously develop neurological dysfunction and spongiform neuropathology. Future investigations of prion diseases using molecular biological and genetic approaches promise to yield much new information about these once enigmatic disorders.     

Convection-Enhanced Delivery of AAV2-PrPshRNA in Prion-Infected Mice

Prion disease is caused by a single pathogenic protein (PrP^Sc), an abnormal conformer of the normal cellular prion protein PrP^C. Depletion of PrP^C in prion knockout mice makes them resistant to prion disease. Thus, gene silencing of the Prnp gene is a promising effective therapeutic approach. Here, we examined adeno-associated virus vector type 2 encoding a short hairpin RNA targeting Prnp mRNA (AAV2-PrP-shRNA) to suppress PrP^C expression both in vitro and in vivo. AAV2-PrP-shRNA treatment suppressed PrP levels and prevented dendritic degeneration in RML-infected brain aggregate cultures. Infusion of AAV2-PrP-shRNA-eGFP into the thalamus of CD-1 mice showed that eGFP was transported to the cerebral cortex via anterograde transport and the overall PrP^C levels were reduced by ∼70% within 4 weeks. For therapeutic purposes, we treated RML-infected CD-1 mice with AAV2-PrP-shRNA beginning at 50 days post inoculation. Although AAV2-PrP-shRNA focally suppressed PrP^Sc formation in the thalamic infusion site by ∼75%, it did not suppress PrP^Sc formation efficiently in other regions of the brain. Survival of mice was not extended compared to the untreated controls. Global suppression of PrP^C in the brain is required for successful therapy of prion diseases.     

Prion protein expression modulates neuronal copper content

The prion protein is a copper (Cu)-binding protein. The abnormal isoform of this protein is associated with the transmissible spongiform encephalopathies or prion diseases. In prion diseases, the prion protein loses its Cu binding capacity. The effect of prion protein expression on the Cu content of the brain was investigated. Transgenic mice, either overexpressing the prion protein or expressing a mutant form lacking the Cu-binding region of the protein, were compared with wild-type mice and mice in which expression of the protein was knocked out. Age-dependent differences in Cu content of the brain were detected. Also, synaptosomal fractions from the brains of the mice showed different Cu content depending on the expression of the prion protein. Mice expressing prion protein, but without the Cu-binding domain showed reduced Cu content. Mice overexpressing the prion protein showed little difference in Cu in the brain compared with wild type but also the prion protein expressed by the mice showed a reduction in the level of Cu bound. These results confirm that prion protein expression modulates the Cu level found at the synapse and this effect is dependent on its Cu binding capacity. Loss of normal Cu binding by the prion protein altered age-related increases in metals in the brain. This may explain why many forms of human prion disease do not develop until late in life.     

Loss of the N-acetylgalactosamine side chain of the GPI-anchor impairs bone formation and brain functions and accelerates the prion disease pathology

Glycosylphosphatidylinositol (GPI) is a posttranslational glycolipid modification of proteins that anchors proteins in lipid rafts on the cell surface. Although some GPI-anchored proteins (GPI-APs), including the prion protein PrP^C, have a glycan side chain composed of N-acetylgalactosamine (GalNAc)−galactose−sialic acid on the core structure of GPI glycolipid, in vivo functions of this GPI-GalNAc side chain are largely unresolved. Here, we investigated the physiological and pathological roles of the GPI-GalNAc side chain in vivo by knocking out its initiation enzyme, PGAP4, in mice. We show that Pgap4 mRNA is highly expressed in the brain, particularly in neurons, and mass spectrometry analysis confirmed the loss of the GalNAc side chain in PrP^C GPI in PGAP4-KO mouse brains. Furthermore, PGAP4-KO mice exhibited various phenotypes, including an elevated blood alkaline phosphatase level, impaired bone formation, decreased locomotor activity, and impaired memory, despite normal expression levels and lipid raft association of various GPI-APs. Thus, we conclude that the GPI-GalNAc side chain is required for in vivo functions of GPI-APs in mammals, especially in bone and the brain. Moreover, PGAP4-KO mice were more vulnerable to prion diseases and died earlier after intracerebral inoculation of the pathogenic prion strains than wildtype mice, highlighting the protective roles of the GalNAc side chain against prion diseases.     

Design and optimization of lentiviral vectors for transfer of GALC expression in Twitcher brain

BACKGROUND: Demyelination in globoid cell leukodystrophy (GLD) is due to a deficiency of galactocerebrosidase (GALC) activity. Up to now, in vivo brain viral gene transfer of GALC showed modest impact on disease development in Twitcher mice, an animal model for GLD. Lentiviral vectors, which are highly efficient to transfer the expression of therapeutic genes in neurons and glial cells, have not been evaluated for direct cerebral therapy in GLD mice. METHODS: Lentiviral vectors containing the untagged cDNA or the hemagglutinin (HA)-tagged cDNA for the full-length mouse GALC sequence were generated and validated in vitro. In vivo therapeutic efficacy of these vectors was evaluated by histology, biochemistry and electrophysiology after transduction of ependymal or subependymal layers in young Twitcher pups. RESULTS: Both GALC lentiviral vectors transduced neurons, oligodendrocytes and astrocytes with efficiencies above 75% and conferred high levels of enzyme activity. GALC accumulated in lysosomes of transduced cells and was also secreted to the extracellular medium. Conditioned GALC medium was able to correct the enzyme deficiency when added to non-transduced Twitcher glial cultures. Mice that received intraventricular injections of GALC vector showed accumulation of GALC in ependymal cells but no diffusion of the enzyme from the ependymal ventricular tree into the cerebral parenchyma. Significant expression of GALC-HA was detected in neuroglioblasts when GALC-HA lentiviral vectors were injected in the subventricular zone of Twitcher mice. Life span and motor conduction in both groups of treated Twitcher mice were not significantly ameliorated. CONCLUSIONS: Lentiviral vectors showed to be efficient for reconstitution of the GALC expression in Twitcher neural cells. GALC was able to accumulate in lysosomes as well as to enter the secretory pathway of lysosomal enzymes, two fundamental aspects for gene therapy of lysosomal storage diseases. Our in vivo results, while showing the capacity of lentiviral vectors to transfer expression of therapeutic GALC in the Twitcher brain, did not limit progression of disease in Twitchers and highlight the need to evaluate other routes of administration.     

Misfolding leads the way to unraveling signaling pathways in the pathophysiology of prion diseases

A misfolded version of the prion protein represents an essential component in the pathophysiology of fatal neurodegenerative prion diseases, which affect humans and animals alike. They may be of sporadic origin, acquired through exogenous introduction of infectious misfolded prion protein, or caused by genetic alterations in the prion protein coding gene. We have recently described a novel pathway linking retention of mutant prion protein in the early secretory pathway to activation p38-MAPK and a neurodegenerative phenotype in transgenic mice. Here we review the consequences that mutations in prion protein have on intracellular transport and stress responses focusing on protein quality control. We also discuss the neurotoxic signaling elicited by the accumulation of mutant prion protein in the endoplasmic reticulum and the Golgi apparatus. Improved knowledge about these processes will help us to better understand complex pathogenesis of prion diseases, a prerequisite for therapeutic strategies.     

Impaired Motor Coordination in Mice Lacking Prion Protein

1. Prion protein (PrP^C) is a host-encoded glycoprotein constitutively expressed on the neuronal cell surface. Accumulation of its protease-resistant isoform is closely related to pathologic changes and prion propagation in the brain tissue of a series of prion diseases. However, the physiological role of PrP^C remains to be elucidated.2. After long-term observation, we noted impaired motor coordination and loss of cerebellar Purkinje cells in the aged mice homozygous for a disrupted PrP gene, a finding which strongly suggests that PrP^C plays a role in the long-term survival of Purkinje cells.3. We also describe the resistance of the PrP null mice to the prion, indicating the requirement of PrP^C for both the development of prion diseases and the prion propagation.     

Therapeutic effect of peripheral administration of an anti‐prion protein antibody on mice infected with prions

It is generally thought that effective treatments for prion diseases need to inhibit prion propagation, protect neuronal tissues and promote functional recovery of degenerated nerve tissues. In addition, such treatments should be effective even when given after clinical onset of the disease and administered via a peripheral route. In this study, the effect of peripheral administration of an anti‐PrP antibody on disease progression in prion‐infected mice was examined. mAb 31C6 was administered via the tail veins of prion‐infected mice at the time of clinical onset (120 days post‐inoculation with the Chandler prion strain) and the distribution of this mAb in the brain and its effect on mouse survival assessed. The antibody was distributed to the cerebellums and thalami of the infected mice and more than half these mice survived longer than mice that had been given a negative control mAb. The level of PrP^Sc in the mAb 31C6‐treated mice was lower than that in mice treated with the negative control mAb and progression of neuropathological lesions in the cerebellum, where the mAb 31C6 was well distributed, appeared to be mitigated. These results suggest that administration of an anti‐PrP mAb through a peripheral route is a candidate for the treatment of prion diseases.     

Decrease of RyR2 in the prion infected cell line and in the brains of the scrapie infected mice models and the patients of human prion diseases

ABSTRACT

The levels of ryanodine receptors (RyRs) are usually increased in the brains of human Alzheimer disease (AD) and AD animal models. To evaluate the underlying alteration of brain RyRs in prion disease, scrapie infected cell line SMB-S15 and its infected mice were tested. RyR2 specific Western blots revealed markedly decreased RyR2 levels both in the cells and in the brains of infected mice. Assays of the brain samples of other scrapie (agents 139A and ME7) infected mice collected at different time-points during incubation period showed time-dependent decreases of RyR2. Immunofluorescent assays (IFA) verified that the expression of RyR2 locates predominantly in cytoplasm of SMB cells and overlapped with the neurons in the brain slices of mice. Furthermore, significant down-regulation of RyR2 was also detected in the postmortem cortical brains of the patients of various types of human prion diseases, including sporadic Creutzfeldt-Jakob disease (sCJD), fatal familial insomnia (FFI) and G114V-genetic CJD. Our data here propose the evidences of remarkably decreased brain RyR2 at terminal stages of both human prion diseases and prion infected rodent models. It also highlights that the therapeutic strategy with antagonist of RyRs in AD may not be suitable for prion disease.     

Efficient and specific down-regulation of prion protein expression by RNAi

Prion diseases are fatal neurodegenerative disorders associated with an abnormal isoform of the PrPc host-encoded protein. Invalidation of the Prnp gene, that encodes PrPc, led to transgenic mice that are viable, apparently healthy, and resistant to challenge by the infectious agent. These results indicated that a down-regulation of the Prnp gene expression is a potential therapeutic approach. In the present report, we demonstrate that RNAi targeted towards the Prnp mRNA can efficiently and highly specifically reduce the level of PrPc in transfected cells. It, thus, indicates that RNAi is an attractive therapeutic approach to fight against prion diseases.     

Utility of intraperitoneal administration as a route of AAV serotype 5 vector-mediated neonatal gene transfer

BACKGROUND: Gene transfer into a fetus or neonate can be a fundamental approach for treating genetic diseases, particularly disorders that have irreversible manifestations in adulthood. Although the potential utility of this technique has been suggested, the advantages of neonatal gene transfer have not been widely investigated. Here, we tested the usefulness of neonatal gene transfer using adeno-associated virus (AAV) vectors by comparing the administration routes and vector doses. METHODS: To determine the optimal administration route, neonates were subjected to intravenous (i.v.) or intraperitoneal (i.p.) injections of AAV5-based vectors encoding the human coagulation factor IX (hfIX) gene, and the dose response was examined. To determine the distribution of transgene expression, vectors encoding lacZ or luciferase (luc) genes were used and assessed by X-gal staining and in vivo imaging, respectively. After the observation period, the vector distribution across tissues was quantified. RESULTS: The factor IX concentration was higher in i.p.-injected mice than in i.v.-injected mice. All transgenes administered by i.p. injection were more efficiently expressed in neonates than in adults. The expression was confined to the peritoneal tissue. Interestingly, a sex-related difference was observed in transgene expression in adults, whereas this difference was not apparent in neonates. CONCLUSIONS: AAV vector administration to neonates using the i.p. route was clearly advantageous in obtaining robust transgene expression. Vector genomes and transgene expression were observed mainly in the peritoneal tissue. These findings indicate the advantages of neonatal gene therapy and would help in designing strategies for gene therapy using AAV vectors.     

Therapy for prion diseases: Insights from the use of RNA interference

Insights into the molecular basis and the temporal evolution of neurotoxicity in prion disease are increasing, and recent work in mice leads to new avenues for targeting treatment of these disorders. Using lentivirally mediated RNA interference (RNAi) against native prion protein (PrP), White et al report the first therapeutic intervention that results in neuronal rescue, prevents symptoms and increases survival in mice with established prion disease.1 Both the target, and the timing, of treatment here are crucial to the effectiveness of this strategy: the formation of the neurotoxic prion agent is prevented at a point when diseased neurons can still be saved from death. But the data also give new insights into the timing of treatment in the context of the pattern of spread of prion infection throughout the brain, with implications for developing the most effective treatments.

This perspective considers developments in the field that led to the rationale for targeting endogenous prion protein (PrP) in prion therapeutics and to the discovery of a window of reversibility of early neuronal damage in prion disease. It introduces RNA interference (RNAi) and its therapeutic use in this context and discusses insights into prion pathogenesis and future treatment strategies and goals. A key concept is targeting the critical brain regions for the spread of prion replication. This may have relevance in other neurodegenerative diseases due to protein misfolding, which recent literature suggests may also propagate throughout the brain in disease-specific patterns.     

A synthetic peptide initiates Gerstmann-Sträussler-Scheinker (GSS) disease in transgenic mice

The molecular basis of the infectious, inherited and sporadic forms of prion diseases is best explained by a conformationally dimorphic protein that can exist in distinct normal and disease-causing isoforms. We identified a 55-residue peptide of a mutant prion protein that can be refolded into at least two distinct conformations. When inoculated intracerebrally into the appropriate transgenic mouse host, 20 of 20 mice receiving the beta-form of this peptide developed signs of central nervous system dysfunction at approximately 360 days, with neurohistologic changes that are pathognomonic of Gerstmann-Str?ussler-Scheinker disease. By contrast, eight of eight mice receiving a non-beta-form of the peptide failed to develop any neuropathologic changes more than 600 days after the peptide injections. We conclude that a chemically synthesized peptide refolded into the appropriate conformation can accelerate or possibly initiate prion disease.     

High incidence of scrapie induced by repeated injections of subinfectious prion doses

To clarify the mechanisms leading to the development of Creutzfeldt-Jakob disease in some recipients of pituitary-derived human growth hormone (hGH), we investigated the effects of repeated injections of low prion doses in mice. The injections were performed, as in hGH-treated children, by a peripheral route at short intervals and for an extended period. Twelve groups of 24 mice were intraperitoneally inoculated one, two, or five times per week for 200 days with 2 x 10(-5) to 2 x 10(-8) dilutions of brain homogenate containing the mouse-adapted C506M3 scrapie strain. Sixteen control mice were injected once a week for 200 days with a 2 x 10(-4) dilution of normal brain homogenate. Of mice injected in a single challenge with a scrapie inoculum of a 2 x 10(-4), 2 x 10(-5), or 2 x 10(-6) dilution, 2/10, 1/10, and 0/10 animals developed scrapie, respectively. Control mice remained healthy. One hundred thirty-five of 135 mice injected with repeated prion doses of a 2 x 10(-5) or 2 x 10(-6) dilution succumbed to scrapie. Of mice injected with repeated scrapie doses of a 2 x 10(-7) or 2 x 10(-8) dilution, 52/59 and 38/67 animals died of scrapie, respectively. A high incidence of scrapie was observed in mice receiving repeated doses at low infectivity, whereas there was no disease in mice that were injected once with the same doses. Repeated injections of low prion doses thus constitute a risk for development of prion disease even if the same total dose inoculated in a single challenge does not induce the disease.     

Dissociation of Prion Protein Amyloid Seeding from Transmission of a Spongiform Encephalopathy

Misfolding and aggregation of proteins are common pathogenic mechanisms of a group of diseases called proteinopathies. The formation and spread of proteinaceous lesions within and between individuals were first described in prion diseases and proposed as the basis of their infectious nature. Recently, a similar “prion-like” mechanism of transmission has been proposed in other neurodegenerative diseases such as Alzheimer''s disease. We investigated if misfolding and aggregation of corrupted prion protein (PrP^TSE) are always associated with horizontal transmission of disease. Knock-in transgenic mice (101LL) expressing mutant PrP (PrP-101L) that are susceptible to disease but do not develop any spontaneous neurological phenotype were inoculated with (i) brain extracts containing PrP^TSE from healthy 101LL mice with PrP plaques in the corpus callosum or (ii) brain extracts from mice overexpressing PrP-101L with neurological disease, severe spongiform encephalopathy, and formation of proteinase K-resistant PrP^TSE. In all instances, 101LL mice developed PrP plaques in the area of inoculation and vicinity in the absence of clinical disease or spongiform degeneration of the brain. Importantly, 101LL mice did not transmit disease on serial passage, ruling out the presence of subclinical infection. Thus, in both experimental models the formation of PrP^TSE is not infectious. These results have implications for the interpretation of tests based on the detection of protein aggregates and suggest that de novo formation of PrP^TSE in the host does not always result in a transmissible prion disease. In addition, these results question the validity of assuming that all diseases due to protein misfolding can be transmitted between individuals.     

Immunological aspects of recombinant adeno-associated virus delivery to the mammalian brain

Recombinant adeno-associated viruses (rAAV) are highly efficient vectors for gene delivery into the central nervous system (CNS). However, host inflammatory and immune responses may play a critical role in limiting the use of rAAV vectors for gene therapy and functional genomic studies in vivo. Here, we evaluated the effect of repeated injections of five rAAV vectors expressing different genetic sequences (coding or noncoding) in a range of combinations into the rat brain. Specifically, we wished to determine whether a specific immune or inflammatory response appeared in response to the vector and/or the transgene protein after repeated injections under conditions of mannitol coinjection. We show that readministration of the same rAAV to the CNS is possible if the interval between the first and second injection is more than 4 weeks. Furthermore, our data demonstrate that rAAV vectors carrying different genetic sequences can be administered at intervals of 2 weeks. Our data therefore suggest that the AAV capsid structure is altered by the vector genetic sequence, such that secondary structures of the single-stranded genome have an impact on the antigenicity of the virus. This study provides guidelines for more rational design of gene transfer studies in the rodent brain and, in addition, suggests the use of repeated administration of rAAV as a viable form of therapy for the treatment of chronic diseases.     

Upregulation of the genes encoding lysosomal hydrolases, a perforin-like protein, and peroxidases in the brains of mice affected with an experimental prion disease

In an attempt to identify the molecules involved in the pathogenesis of prion diseases, we performed cDNA subtraction on the brain tissues of mice affected with an experimental prion disease and the unaffected control. The genes identified as being upregulated in the prion-affected brain tissue included those encoding a series of lysosomal hydrolases (lysozyme M and both isoforms of beta-N-acetylhexosaminidase), a perforin-like protein (macrophage proliferation-specific gene-1 [MPS-1]), and an oxygen radical scavenger (peroxiredoxin). Dramatic increases in the expression level occurred at between 12 and 16 weeks after intracerebral inoculation of the prion, coinciding with the onset of spongiform degeneration. The proteinase K-resistant prion protein (PrP(Sc)) became detectable by immunoblotting well before 12 weeks, suggesting a causal relationship between this and the gene activation. Immunohistochemistry paired with in situ hybridization on sections of the affected brain tissue revealed that expression of the peroxiredoxin gene was detectable only in astrocytes and was noted throughout the affected brain tissue. On the other hand, the genes for the lysosomal hydrolases and MPS-1 were overexpressed exclusively by microglia, which colocalized with the spongiform morphological changes. A crucial role for microglia in the spongiform degeneration by their production of neurotoxic substances, and possibly via the aberrant activation of the lysosomal system, would have to be considered.