Bruce Pulford

Liposome-siRNA-Peptide Complexes Cross the Blood-Brain Barrier and Significantly Decrease PrPC on Neuronal Cells and PrPRES in Infected Cell Cultures

Background Recent advances toward an effective therapy for prion diseases employ RNA interference to suppress PrPC expression and subsequent prion neuropathology, exploiting the phenomenon that disease severity and progression correlate with host PrPC expression levels. However, delivery of lentivirus encoding PrP shRNA has demonstrated only modest efficacy in vivo. Methodology/Principal Findings Here we describe a new siRNA delivery system incorporating a small peptide that binds siRNA and acetylcholine receptors (AchRs), acting as a molecular messenger for delivery to neurons, and cationic liposomes that protect siRNA-peptide complexes from serum degradation. Conclusions/Significance Liposome-siRNA-peptide complexes (LSPCs) delivered PrP siRNA specifically to AchR-expressing cells, suppressed PrPC expression and eliminated PrPRES formation in vitro. LSPCs injected intravenously into mice resisted serum degradation and delivered PrP siRNA throughout the brain to AchR and PrPC-expressing neurons. These data promote LSPCs as effective vehicles for delivery of PrP and other siRNAs specifically to neurons to treat prion and other neuropathological diseases.

Detection of protease-resistant cervid prion protein in water from a CWD-endemic area.

Chronic wasting disease (CWD) is the only known transmissible spongiform encephalopathy affecting free-ranging wildlife. Although the exact mode of natural transmission remains unknown, substantial evidence suggests that prions can persist in the environment, implicating components thereof as potential prion reservoirs and transmission vehicles. CWD-positive animals may contribute to environmental prion load via decomposing carcasses and biological materials including saliva, blood, urine and feces. Sensitivity limitations of conventional assays hamper evaluation of environmental prion loads in soil and water. Here we show the ability of serial protein misfolding cyclic amplification (sPMCA) to amplify a 1.3 x 10-7 dilution of CWD-infected brain homogenate spiked into water samples, equivalent to approximately 5 x 107 protease resistant cervid prion protein (PrPCWD) monomers. We also detected PrPCWD in one of two environmental water samples from a CWD endemic area collected at a time of increased water runoff from melting winter snow pack, as well as in water samples obtained concurrently from the flocculation stage of water processing by the municipal water treatment facility. Bioassays indicated that the PrPCWD detected was below infectious levels. These data demonstrate detection of very low levels of PrPCWD in the environment by sPMCA and suggest persistence and accumulation of prions in the environment that may promote CWD transmission.

Uptake of Circulating Insulin-Like Growth Factors (IGFs) into Cerebrospinal Fluid Appears to Be Independent of the IGF Receptors as Well as IGF-Binding Proteins1

Peripheral administration of human insulin-like growth factor (hIGF) results in both uptake of hIGF into the cerebrospinal fluid (CSF) and amelioration of brain injury. We tested the hypotheses that IGF uptake into CSF is independent of IGF receptors and IGF-binding proteins (IGFBP). Adult rats were injected sc with various concentrations of hIGF-I or structural analogs, and serum and CSF were withdrawn for assay 90 min later. An enzyme-linked immunoassay was used that detected immunoreactive hIGF-I and its analogs, but not rat IGF-I, IGF-II, or insulin. Plasma hIGF-I levels increased linearly (r = 0.97) with hIGF-I dose between 25-300 microgram/rat. By contrast, uptake into CSF reached saturation above 100 microgram, suggesting carrier-mediated uptake. hIGF-II reduced the uptake of hIGF-I into CSF (P < 0.02). Des(1-3)hIGF-I is a hIGF-I analog missing the N-terminal tripeptide, resulting in greatly reduced affinity for IGFBP-1, -3, -4, and -5. Nevertheless, des(1-3)hIGF-I was taken up into CSF. [Leu(24)]hIGF-I and [Leu(60)]hIGF-I have 20- to 85-fold reduced affinity for the type I IGF receptor, yet both were taken up into CSF in amounts similar to hIGF-I. In addition, hIGF-I and des(1-3)hIGF-I were taken up into CSF, although binding to the type II receptor is extremely weak. These data suggest that uptake of circulating IGF-I into CSF is independent of the type I or II IGF receptors as well as IGF sequestration to IGFBP-1, -3, -4, or -5.

In vitro strain adaptation of CWD prions by serial protein misfolding cyclic amplification

We used serial protein misfolding cyclic amplification (sPMCA) to amplify the D10 strain of CWD prions in a linear relationship over two logs of D10 dilutions. The resultant PMCA-amplified D10 induced terminal TSE disease in CWD-susceptible Tg(cerPrP)1536 mice with a survival time approximately 80 days shorter than the original D10 inoculum, similar to that produced by in vivo sub-passage of D10 in Tg(cerPrP)1536 mice. Both in vitro-amplified and mouse-passaged D10 produced brain lesion profiles, glycoform ratios and conformational stabilities significantly different than those produced by the original D10 inoculum in Tg(cerPrP)1536 mice. These findings demonstrate that sPMCA can amplify and adapt prion strains in vitro as effectively and much more quickly than in vivo strain adaptation by mouse passage. Thus sPMCA may represent a powerful tool to assess prion strain adaptation and species barriers in vitro.

DETECTION OF PrP CWD IN FECES FROM NATURALLY EXPOSED ROCKY MOUNTAIN ELK (CERVUS ELAPHUS NELSONI) USING PROTEIN MISFOLDING CYCLIC AMPLIFICATION

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting captive and free-ranging cervids. Currently, tests for CWD in live animals involve relatively invasive procedures to collect lymphoid tissue biopsies and examine them for CWD-associated, protease-resistant cervid prion protein (PrPCWD) detected by immunohistochemistry (IHC). We adapted an ultrasensitive prion detection system, protein misfolding cyclic amplification (PMCA), to detect PrPCWD in Rocky Mountain elk (Cervus elaphus nelsoni) feces. Our PMCA reproducibly detected a 1.2×107 dilution of PrPCWD (a 10% infected brain homogenate diluted 1.2×106-fold into 10% fecal homogenates), equivalent to approximately 100 pg of PrPCWD/g of feces. We developed a semiquantitative scoring system based on the first PMCA round at which PrPCWD was detected and fit a nonlinear regression curve to our serial dilutions to correlate PMCA scores with known PrPCWD concentrations. We used this PMCA scoring system to detect PrPCWD and estimate its concentration in feces from free-ranging elk from Rocky Mountain National Park, Colorado. We compared our results to PrPCWD IHC of rectoanal mucosa-associated lymphoid tissue and obex from the same animals. The PMCA successfully detected PrPCWD in feces from elk that were positive by IHC, with estimated prion loads from 100 to 5,000 pg PrPCWD/g of feces. These data show for the first time PrPCWD in feces from naturally exposed free-ranging elk and demonstrate the potential of PMCA as a new, noninvasive CWD diagnostic tool to complement IHC.

Peripherally administered insulin-like growth factor-I preserves hindlimb reflex and spinal cord noradrenergic circuitry following a central nervous system lesion in rats.

The blood-central nervous system-barrier (B-CNS-B) is widely considered a significant impediment to the use of protein neurotrophic factors for the treatment of brain diseases and disorders. In this study, we tested the hypothesis that systemic administration of insulin-like growth factor I (IGF-I) can ameliorate functional damage to the central nervous system. Intracisternal injection of 6-hydroxydopamine (6-OHDA) normally results in loss of both the descending spinal cord noradrenergic (NA) fibers and the hindlimb withdrawal reflex. Ten minutes after 6-OHDA or solvent injection, 1 week duration osmotic minipumps containing IGF-I or vehicle were implanted subcutaneously in the mid-back of adult rats. Three weeks post-surgery, the maximum stimulus-evoked withdrawal force of the hindlimb was measured. This withdrawal reflex was significantly reduced in 6-OHDA lesioned vs. nonlesioned rats (P <.0002). The mean maximum reflex force was significantly larger in IGF-I vs. vehicle-treated lesioned rats (P < 0.008). Following reflex testing, serial sections of the spinal cord were taken through the lumbar enlargement containing the motoneurons mediating the hindlimb reflexes. The interspersed NA axons and their bead-like varicosities were stained with an anti-dopamine-beta-hydroxylase antibody. The mean number of NA varicosities per unit area in the ventral horn was profoundly reduced in lesioned vs. nonlesioned rats (P < 0.0002), but significant numbers (51%) were retained in lesioned rats treated with IGF-I vs. vehicle (P < 0.02). These data suggest that blood-borne IGF-I preserves both reflex function and spinal cord circuitry following injury to NA axons and that the blood-CNS fluid barriers may not be an impediment for IGF-I entry into the CNS.

Incunabular Immunological Events in Prion Trafficking

While prions probably interact with the innate immune system immediately following infection, little is known about this initial confrontation. Here we investigated incunabular events in lymphotropic and intranodal prion trafficking by following highly enriched, fluorescent prions from infection sites to draining lymph nodes. We detected biphasic lymphotropic transport of prions from the initial entry site upon peripheral prion inoculation. Prions arrived in draining lymph nodes cell autonomously within two hours of intraperitoneal administration. Monocytes and dendritic cells (DCs) required Complement for optimal prion delivery to lymph nodes hours later in a second wave of prion trafficking. B cells constituted the majority of prion-bearing cells in the mediastinal lymph node by six hours, indicating intranodal prion reception from resident DCs or subcapsulary sinus macrophages or directly from follicular conduits. These data reveal novel, cell autonomous prion lymphotropism, and a prominent role for B cells in intranodal prion movement.

Genetic Depletion of Complement Receptors CD21/35 Prevents Terminal Prion Disease in a Mouse Model of Chronic Wasting Disease

The complement system has been shown to facilitate peripheral prion pathogenesis. Mice lacking complement receptors CD21/35 partially resist terminal prion disease when infected i.p. with mouse-adapted scrapie prions. Chronic wasting disease (CWD) is an emerging prion disease of captive and free-ranging cervid populations that, similar to scrapie, has been shown to involve the immune system, which probably contributes to their relatively facile horizontal and environmental transmission. In this study, we show that mice overexpressing the cervid prion protein and susceptible to CWD (Tg(cerPrP)5037 mice) but lack CD21/35 expression completely resist clinical CWD upon peripheral infection. CD21/35-deficient Tg5037 mice exhibit greatly impaired splenic prion accumulation and replication throughout disease, similar to CD21/35-deficient murine prion protein mice infected with mouse scrapie. TgA5037;CD21/35−/− mice exhibited little or no neuropathology and deposition of misfolded, protease-resistant prion protein associated with CWD. CD21/35 translocate to lipid rafts and mediates a strong germinal center response to prion infection that we propose provides the optimal environment for prion accumulation and replication. We further propose a potential role for CD21/35 in selecting prion quasi-species present in prion strains that may exhibit differential zoonotic potential compared with the parental strains.

Amyloid-β and proinflammatory cytokines utilize a prion protein-dependent pathway to activate NADPH oxidase and induce cofilin-actin rods in hippocampal neurons.

Neurites of neurons under acute or chronic stress form bundles of filaments (rods) containing 1∶1 cofilin∶actin, which impair transport and synaptic function. Rods contain disulfide cross-linked cofilin and are induced by treatments resulting in oxidative stress. Rods form rapidly (5–30 min) in >80% of cultured hippocampal or cortical neurons treated with excitotoxic levels of glutamate or energy depleted (hypoxia/ischemia or mitochondrial inhibitors). In contrast, slow rod formation (50% of maximum response in ∼6 h) occurs in a subpopulation (∼20%) of hippocampal neurons upon exposure to soluble human amyloid-β dimer/trimer (Aβd/t) at subnanomolar concentrations. Here we show that proinflammatory cytokines (TNFα, IL-1β, IL-6) also induce rods at the same rate and within the same neuronal population as Aβd/t. Neurons from prion (PrPC)-null mice form rods in response to glutamate or antimycin A, but not in response to proinflammatory cytokines or Aβd/t. Two pathways inducing rod formation were confirmed by demonstrating that NADPH-oxidase (NOX) activity is required for prion-dependent rod formation, but not for rods induced by glutamate or energy depletion. Surprisingly, overexpression of PrPC is by itself sufficient to induce rods in over 40% of hippocampal neurons through the NOX-dependent pathway. Persistence of PrPC-dependent rods requires the continuous activity of NOX. Removing inducers or inhibiting NOX activity in cells containing PrPC-dependent rods causes rod disappearance with a half-life of about 36 min. Cofilin-actin rods provide a mechanism for synapse loss bridging the amyloid and cytokine hypotheses for Alzheimer disease, and may explain how functionally diverse Aβ-binding membrane proteins induce synaptic dysfunction.

Complement protein C3 exacerbates prion disease in a mouse model of chronic wasting disease

Accumulating evidence shows a critical role of the complement system in facilitating attachment of prions to both B cells and follicular dendritic cells and assisting in prion replication. Complement activation intensifies disease in prion-infected animals, and elimination of complement components inhibits prion accumulation, replication and pathogenesis. Chronic wasting disease (CWD) is a highly infectious prion disease of captive and free-ranging cervid populations that utilizes the complement system for efficient peripheral prion replication and most likely efficient horizontal transmission. Here we show that complete genetic or transient pharmacological depletion of C3 prolongs incubation times and significantly delays splenic accumulation in a CWD transgenic mouse model. Using a semi-quantitative prion amplification scoring system we show that C3 impacts disease progression in the early stages of disease by slowing the rate of prion accumulation and/or replication. The delayed kinetics in prion replication correlate with delayed disease kinetics in mice deficient in C3. Taken together, these data support a critical role of C3 in peripheral CWD prion pathogenesis.