Kimberly Meade-White

Mutations associated with neutropenia in dogs and humans disrupt intracellular transport of neutrophil elastase

Cyclic hematopoiesis is a stem cell disease in which the number of neutrophils and other blood cells oscillates in weekly phases. Autosomal dominant mutations of ELA2, encoding the protease neutrophil elastase, found in lysosome-like granules, cause cyclic hematopoiesis and most cases of the pre-leukemic disorder severe congenital neutropenia (SCN; ref. 3) in humans. Over 20 different mutations of neutrophil elastase have been identified, but their consequences are elusive, because they confer no consistent effects on enzymatic activity. The similar autosomal recessive disease of dogs, canine cyclic hematopoiesis, is not caused by mutations in ELA2 (data not shown). Here we show that homozygous mutation of the gene encoding the dog adaptor protein complex 3 (AP3) β-subunit, directing trans-Golgi export of transmembrane cargo proteins to lysosomes, causes canine cyclic hematopoiesis. C-terminal processing of neutrophil elastase exposes an AP3 interaction signal responsible for redirecting neutrophil elastase trafficking from membranes to granules. Disruption of either neutrophil elastase or AP3 perturbs the intracellular trafficking of neutrophil elastase. Most mutations in ELA2 that cause human cyclic hematopoiesis prevent membrane localization of neutrophil elastase, whereas most mutations in ELA2 that cause SCN lead to exclusive membrane localization.

Fatal Transmissible Amyloid Encephalopathy: A New Type of Prion Disease Associated with Lack of Prion Protein Membrane Anchoring

Prion diseases are fatal neurodegenerative diseases of humans and animals characterized by gray matter spongiosis and accumulation of aggregated, misfolded, protease-resistant prion protein (PrPres). PrPres can be deposited in brain in an amyloid-form and/or non-amyloid form, and is derived from host-encoded protease-sensitive PrP (PrPsen), a protein normally anchored to the plasma membrane by glycosylphosphatidylinositol (GPI). Previously, using heterozygous transgenic mice expressing only anchorless PrP, we found that PrP anchoring to the cell membrane was required for typical clinical scrapie. However, in the present experiments, using homozygous transgenic mice expressing two-fold more anchorless PrP, scrapie infection induced a new fatal disease with unique clinical signs and altered neuropathology, compared to non-transgenic mice expressing only anchored PrP. Brain tissue of transgenic mice had high amounts of infectivity, and histopathology showed dense amyloid PrPres plaque deposits without gray matter spongiosis. In contrast, infected non-transgenic mice had diffuse non-amyloid PrPres deposits with significant gray matter spongiosis. Brain graft studies suggested that anchored PrPsen expression was required for gray matter spongiosis during prion infection. Furthermore, electron and light microscopic studies in infected transgenic mice demonstrated several pathogenic processes not seen in typical prion disease, including cerebral amyloid angiopathy and ultrastructural alterations in perivascular neuropil. These findings were similar to certain human familial prion diseases as well as to non-prion human neurodegenerative diseases, such as Alzheimers disease.

Susceptibilities of nonhuman primates to chronic wasting disease.

A species barrier may protect humans from this disease.

Subclinical Scrapie Infection in a Resistant Species: Persistence, Replication, and Adaptation of Infectivity during Four Passages

Cross-species infection with transmissible spongiform encephalopathy agents may lead to subclinical infection and to adaptation of the infection to new species. This is of particular concern for the millions of people possibly exposed to bovine spongiform encephalopathy (BSE) by consumption of BSE-infected beef. Subclinical infection was studied by making 4 serial passages of hamster scrapie agent (263K) in mice. At each step, infectivity was followed by inoculation of hamsters and mice. Subclinical infection was demonstrated either by detection of abnormal protease-resistant prion protein (PrP-res) or in the absence of PrP-res by detection of infectivity. Replication and adaptation of hamster infectivity in mice was shown in year 2 after initial mouse passage. In third and fourth passages, dual-tropic, mouse-tropic, and hamster-tropic infectivity was found in different animals. In some cases infectivity similar to the original 263K hamster scrapie strain was found after 2 or 3 serial mouse passages totaling 1200-1550 days.

A novel notch protein, N2N, targeted by neutrophil elastase and implicated in hereditary neutropenia.

ABSTRACT Mutations in ELA2, encoding the human serine protease neutrophil elastase, cause cyclic and severe congenital neutropenia, and recent evidence indicates that the mutations alter the membrane trafficking of neutrophil elastase. These disorders feature impaired bone marrow production of neutrophils along with excess monocytes—terminally differentiated lineages corresponding to the two alternative fates of myeloid progenitor cells. We utilized a modified yeast two-hybrid system and identified a new, widely expressed gene, N2N, whose product is homologous to Notch2, that interacts with neutrophil elastase. N2N is a 36-kDa protein distributed throughout the cell and secreted. Its amino-terminal sequence consists of several EGF repeats identical to those of the extracellular region of Notch2, and its carboxyl terminus contains a unique 24-residue domain required for interaction with neutrophil elastase. Neutrophil elastase cleaves N2N within EGF repeats in vitro and in living cells, but the C-terminal domain retards proteolysis. In vitro, N2N represses transcriptional activities of Notch proteins. Disease-causing mutations of neutrophil elastase disrupt the interaction with N2N, impair proteolysis of N2N and Notch2, and interfere with Notch2 signaling, suggesting defective proteolysis of an inhibitory form of Notch as an explanation for the alternate switching of cell fates characteristic of hereditary neutropenia.

Resistance to Chronic Wasting Disease in Transgenic Mice Expressing a Naturally Occurring Allelic Variant of Deer Prion Protein

ABSTRACT Prion protein (PrP) is a required factor for susceptibility to transmissible spongiform encephalopathy or prion diseases. In transgenic mice, expression of prion protein (PrP) from another species often confers susceptibility to prion disease from that donor species. For example, expression of deer or elk PrP in transgenic mice has induced susceptibility to chronic wasting disease (CWD), the prion disease of cervids. In the current experiments, transgenic mice expressing two naturally occurring allelic variants of deer PrP with either glycine (G) or serine (S) at residue 96 were found to differ in susceptibility to CWD infection. G96 mice were highly susceptible to infection, and disease appeared starting as early as 160 days postinfection. In contrast, S96 mice showed no evidence of disease or generation of disease-associated protease-resistant PrP (PrPres) over a 600-day period. At the time of clinical disease, G96 mice showed typical vacuolar pathology and deposition of PrPres in many brain regions, and in some individuals, extensive neuronal loss and apoptosis were noted in the hippocampus and cerebellum. Extraneural accumulation of PrPres was also noted in spleen and intestinal tissue of clinically ill G96 mice. These results demonstrate the importance of deer PrP polymorphisms in susceptibility to CWD infection. Furthermore, this deer PrP transgenic model is the first to demonstrate extraneural accumulation of PrPres in spleen and intestinal tissue and thus may prove useful in studies of CWD pathogenesis and transmission by oral or other natural routes of infection.

A synthetic consensus anti–spike protein DNA vaccine induces protective immunity against Middle East respiratory syndrome coronavirus in nonhuman primates

A consensus MERS spike protein synthetic DNA vaccine can induce protective responses against viral challenge. Emerging vaccines Public outcry drives vaccine research during outbreaks of emerging infectious disease, but public support for vaccine development dries up when the outbreaks are resolved, frequently leaving promising vaccine candidates sitting on the shelf. DNA vaccines, with their potential for rapid large-scale production, may help overcome this hurdle. Muthumani et al. report the development of a synthetic DNA vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) that induces neutralizing antibodies in mice, macaques, and camels—natural hosts of MERS-CoV. Indeed, macaques vaccinated with this DNA vaccine were protected from viral challenge. These promising results support further development of DNA vaccines for emerging infections. First identified in 2012, Middle East respiratory syndrome (MERS) is caused by an emerging human coronavirus, which is distinct from the severe acute respiratory syndrome coronavirus (SARS-CoV), and represents a novel member of the lineage C betacoronoviruses. Since its identification, MERS coronavirus (MERS-CoV) has been linked to more than 1372 infections manifesting with severe morbidity and, often, mortality (about 495 deaths) in the Arabian Peninsula, Europe, and, most recently, the United States. Human-to-human transmission has been documented, with nosocomial transmission appearing to be an important route of infection. The recent increase in cases of MERS in the Middle East coupled with the lack of approved antiviral therapies or vaccines to treat or prevent this infection are causes for concern. We report on the development of a synthetic DNA vaccine against MERS-CoV. An optimized DNA vaccine encoding the MERS spike protein induced potent cellular immunity and antigen-specific neutralizing antibodies in mice, macaques, and camels. Vaccinated rhesus macaques seroconverted rapidly and exhibited high levels of virus-neutralizing activity. Upon MERS viral challenge, all of the monkeys in the control-vaccinated group developed characteristic disease, including pneumonia. Vaccinated macaques were protected and failed to demonstrate any clinical or radiographic signs of pneumonia. These studies demonstrate that a consensus MERS spike protein synthetic DNA vaccine can induce protective responses against viral challenge, indicating that this strategy may have value as a possible vaccine modality against this emerging pathogen.

Prion-Induced Amyloid Heart Disease with High Blood Infectivity in Transgenic Mice

We investigated extraneural manifestations in scrapie-infected transgenic mice expressing prion protein lacking the glycophosphatydylinositol membrane anchor. In the brain, blood, and heart, both abnormal protease-resistant prion protein (PrPres) and prion infectivity were readily detected by immunoblot and by inoculation into nontransgenic recipients. The titer of infectious scrapie in blood plasma exceeded 107 50% infectious doses per milliliter. The hearts of these transgenic mice contained PrPres-positive amyloid deposits that led to myocardial stiffness and cardiac disease.

Lower specific infectivity of protease-resistant prion protein generated in cell-free reactions

Prions are unconventional infectious agents that cause transmissible spongiform encephalopathy (TSE) diseases, or prion diseases. The biochemical nature of the prion infectious agent remains unclear. Previously, using a protein misfolding cyclic amplification (PMCA) reaction, infectivity and disease-associated protease-resistant prion protein (PrPres) were both generated under cell-free conditions, which supported a nonviral hypothesis for the agent. However, these studies lacked comparative quantitation of both infectivity titers and PrPres, which is important both for biological comparison with in vivo-derived infectivity and for excluding contamination to explain the results. Here during four to eight rounds of PMCA, end-point dilution titrations detected a >320-fold increase in infectivity versus that in controls. These results provide strong support for the hypothesis that the agent of prion infectivity is not a virus. PMCA-generated samples caused the same clinical disease and neuropathology with the same rapid incubation period as the input brain-derived scrapie samples, providing no evidence for generation of a new strain in PMCA. However, the ratio of the infectivity titer to the amount of PrPres (specific infectivity) was much lower in PMCA versus brain-derived samples, suggesting the possibility that a substantial portion of PrPres generated in PMCA might be noninfectious.

Crucial Role for Prion Protein Membrane Anchoring in the Neuroinvasion and Neural Spread of Prion Infection

ABSTRACT In nature prion diseases are usually transmitted by extracerebral prion infection, but clinical disease results only after invasion of the central nervous system (CNS). Prion protein (PrP), a host-encoded glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein, is necessary for prion infection and disease. Here, we investigated the role of the anchoring of PrP on prion neuroinvasion by studying various inoculation routes in mice expressing either anchored or anchorless PrP. In control mice with anchored PrP, intracerebral or sciatic nerve inoculation resulted in rapid CNS neuroinvasion and clinical disease (154 to 156 days), and after tongue, ocular, intravenous, or intraperitoneal inoculation, CNS neuroinvasion was only slightly slower (193 to 231 days). In contrast, in anchorless PrP mice, these routes resulted in slow and infrequent CNS neuroinvasion. Only intracerebral inoculation caused brain PrPres, a protease-resistant isoform of PrP, and disease in both types of mice. Thus, anchored PrP was an essential component for the rapid neural spread and CNS neuroinvasion of prion infection.