Neuronal cells derived from human induced pluripotent stem cells as a functional tool of melanocortin system.

BACKGROUND: The preparation of human neurons derived from human induced pluripotent stem (iPS) cells can serve as a potential tool for evaluating the physiological and pathophysiological properties of human neurons and for drug development. METHODS: In the present study, the functional activity in neuronal cells differentiated from human iPS cells was observed. RESULTS: The differentiated cells expressed mRNAs for classical neuronal markers (microtubule-associated protein 2, ß-tubulin III, calbindin 1, synaptophysin and postsynaptic density protein 95) and for subunits of various excitatory and inhibitory transmitters (NR1, NR2A, NR2B, GABAA α1). Moreover, the differentiated cells expressed neuropeptides and receptors which are predominantly present in the hypothalamus. The expression of mRNA for preopiomelanocortin, agouti-related protein (AgRP), melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R) increased in culture with a peak on Day 30 which subsequently decreased at Day 45. Immunoreactivities for MC3R and MC4R were also observed in cells differentiated from human iPS cells. Application of a potent agonist for MC3R and MC4R, [Nle4, D-Phe7]-α-melanocyte-stimulating hormone, significantly increased intracellular cAMP levels, but this was suppressed by AgRP (83-132) and SHU9119. CONCLUSIONS: These findings offer the possibility for drug developments using neurons differentiated from normal or disease-associated human iPS cells.

Hippocampal administration of chondroitinase ABC increases plaque-adjacent synaptic marker and diminishes amyloid burden in aged APPswe/PS1dE9 mice.

INTRODUCTION: Substantial data has shown that the lectican group of chondroitin sulfate proteoglycans are involved in inhibition of axonal plasticity in response to injury in the central nervous system. Increasing evidence indicates that lecticans may also play a role in synaptic plasticity related to memory, especially associated with aging. A recent study has shown that lectican expression is elevated at a young age in the APPswe/PS1dE9 mouse model and Alzheimer's disease (AD) and hippocampal treatment with chondroitinase ABC reversed a loss of contextual fear memory and restored long-term potentiation. The purpose of this study was to examine the presence of a synaptic lectican in AD tissue, determine if amyloid-ß (Aß) binds to lecticans purified from brain tissue, and examine how treatment of the same AD model with chondroitinase ABC would influence plaque burden and the density of the synaptic marker synaptophysin around plaques. RESULTS: In human superior frontal gyrus, levels of the brain-specific lectican, brevican, were significantly elevated in AD compared to non-cognitively impaired subjects, with a trend toward an increase in tissue from subjects with mild cognitive impairment. In vitro immunoprecipitation studies showed that brevican binds to oligomeric and fibrillar Aß1-42, and less so to monomeric Aß1-42. Intrahippocampal injection of 15 months APPswe/PS1dE9 mice with chondroitinase ABC resulted in a reduction of Aß burden in the stratum lacunosum moleculare and a reversal of the loss of synaptic density surrounding plaques in the same region. CONCLUSIONS: It is possible that lecticans, particularly brevican, inhibit synaptic plasticity in this model of AD. Since the hippocampus undergoes changes in synaptic plasticity early in the disease process, it could be possible that removal of lecticans or inhibition of their signaling pathways could prolong plasticity in patients early in the disease process, and delay cognitive decline of AD progression.

ADAMTS expression and function in central nervous system injury and disorders.

The components of the adult extracellular matrix in the central nervous system form a lattice-like structure that is deposited as perineuronal nets, around axon initial segments and as synapse-associated matrix. An abundant component of this matrix is the lecticans, chondroitin sulfate-bearing proteoglycans that are the major substrate for several members of the ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) family. Since lecticans are key regulators of neural plasticity, ADAMTS cleavage of lecticans would likely also contribute to neuroplasticity. Indeed, many studies have examined the neuroplastic contribution of the ADAMTSs to damage and recovery after injury and in central nervous system disease. Much of this data supports a role for the ADAMTSs in recovery and repair following spinal cord injury by stimulating axonal outgrowth after degradation of a glial scar and improving synaptic plasticity following seizure-induced neural damage in the brain. The action of the ADAMTSs in chronic diseases of the central nervous system appears to be more complex and less well-defined. Increasing evidence indicates that lecticans participate in synaptic plasticity in neurodegenerative disease states. It will be interesting to examine how ADAMTS expression and action would affect the progression of these diseases.

Canonical transient receptor channel 5 (TRPC5) and TRPC1/4 contribute to seizure and excitotoxicity by distinct cellular mechanisms.

Seizures are the manifestation of highly synchronized burst firing of a large population of cortical neurons. Epileptiform bursts with an underlying plateau potential in neurons are a cellular correlate of seizures. Emerging evidence suggests that the plateau potential is mediated by neuronal canonical transient receptor potential (TRPC) channels composed of members of the TRPC1/4/5 subgroup. We previously showed that TRPC1/4 double-knockout (DKO) mice lack epileptiform bursting in lateral septal neurons and exhibit reduced seizure-induced neuronal cell death, but surprisingly have unaltered pilocarpine-induced seizures. Here, we report that TRPC5 knockout (KO) mice exhibit both significantly reduced seizures and minimal seizure-induced neuronal cell death in the hippocampus. Interestingly, epileptiform bursting induced by agonists for metabotropic glutamate receptors in the hippocampal CA1 area is unaltered in TRPC5 KO mice, but is abolished in TRPC1 KO and TRPC1/4 DKO mice. In contrast, long-term potentiation is greatly reduced in TRPC5 KO mice, but is normal in TRPC1 KO and TRPC1/4 DKO mice. The distinct changes from these knockouts suggest that TRPC5 and TRPC1/4 contribute to seizure and excitotoxicity by distinct cellular mechanisms. Furthermore, the reduced seizure and excitotoxicity and normal spatial learning exhibited in TRPC5 KO mice suggest that TRPC5 is a promising novel molecular target for new therapy.

Selective decline of synaptic protein levels in the frontal cortex of female mice deficient in the extracellular metalloproteinase ADAMTS1.

The chondroitin sulfate-bearing proteoglycans, also known as lecticans, are a major component of the extracellular matrix (ECM) in the central nervous system and regulate neural plasticity. Growing evidence indicates that endogenous, extracellular metalloproteinases that cleave lecticans mediate neural plasticity by altering the structure of ECM aggregates. The bulk of this in vivo data examined the matrix metalloproteinases, but another metalloproteinase family that cleaves lecticans, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), modulates structural plasticity in vitro, although few in vivo studies have tested this concept. Thus, the purpose of this study was to examine the neurological phenotype of a mouse deficient in ADAMTS1. Adamts1 mRNA was absent in the ADAMTS1 null mouse frontal cortex, but there was no change in the abundance or proteolytic processing of the prominent lecticans brevican and versican V2. However, there was a marked increase in the perinatal lectican neurocan in juvenile ADAMTS1 null female frontal cortex. More prominently, there were declines in synaptic protein levels in the ADAMTS1 null female, but not male, frontal cortex beginning at postnatal day 28. These synaptic marker declines did not affect learning or memory in the adult female ADAMTS1 null mice when tested with the radial-arm water maze. These results indicate that in vivo Adamts1 knockout leads to sexual dimorphism in frontal cortex synaptic protein levels. Since changes in lectican abundance and proteolytic processing did not accompany the synaptic protein declines, ADAMTS1 may play a nonproteolytic role in regulating neural plasticity.

Altered synaptic marker abundance in the hippocampal stratum oriens of Ts65Dn mice is associated with exuberant expression of versican.

DS (Down syndrome), resulting from trisomy of chromosome 21, is the most common cause of genetic mental retardation; however, the molecular mechanisms underlying the cognitive deficits are poorly understood. Growing data indicate that changes in abundance or type of CSPGs (chondroitin sulfate proteoglycans) in the ECM (extracellular matrix) can influence synaptic structure and plasticity. The purpose of this study was to identify changes in synaptic structure in the hippocampus in a model of DS, the Ts65Dn mouse, and to determine the relationship to proteoglycan abundance and/or cleavage and cognitive disability. We measured synaptic proteins by ELISA and changes in lectican expression and processing in the hippocampus of young and old Ts65Dn mice and LMCs (littermate controls). In young (5 months old) Ts65Dn hippocampal extracts, we found a significant increase in the postsynaptic protein PSD-95 (postsynaptic density 95) compared with LMCs. In aged (20 months old) Ts65Dn hippocampus, this increase was localized to hippocampal stratum oriens extracts compared with LMCs. Aged Ts65Dn mice exhibited impaired hippocampal-dependent spatial learning and memory in the RAWM (radial-arm water maze) and a marked increase in levels of the lectican versican V2 in stratum oriens that correlated with the number of errors made in the final RAWM block. Ts65Dn stratum oriens PNNs (perineuronal nets), an extension of the ECM enveloping mostly inhibitory interneurons, were dispersed over a larger area compared with LMC mice. Taken together, these data suggest a possible association with alterations in the ECM and inhibitory neurotransmission in the Ts65Dn hippocampus which could contribute to cognitive deficits.

Trafficking CD11b-positive blood cells deliver therapeutic genes to the brain of amyloid-depositing transgenic mice.

A major question for gene therapy in brain concerns methods to administer therapeutic genes in a uniform manner over major portions of the brain. A second question in neuroimmunology concerns the extent to which monocytes migrate to the CNS in degenerative disorders. Here we show that CD11b+ cells (largely monocytes) isolated from the bone marrow of GFP (green fluorescent protein)-expressing donors spontaneously home to compacted amyloid plaques in the brain. Injections of these cells as a single pulse show a rapid clearance from circulation (90 min half-life) and tissue residence half-lives of approximately 3 d. The uptake into brain was minimal in nontransgenic mice. In transgenic mice containing amyloid deposits, uptake was dramatically increased and associated with a corresponding decrease in monocyte uptake into peripheral organs compared to nontransgenic littermates. Twice weekly infusions of the CD11b+ bone marrow cells transfected with a genetically engineered form of the protease neprilysin completely arrest amyloid deposition in an aggressively depositing transgenic model. Exploiting the natural homing properties of peripherally derived blood cells to deliver therapeutic genes has the advantages of access to the entire CNS, expression largely restricted to sites of injury, low risk of immune reactivity, and fading of expression if adverse reactions are encountered. These observations support the feasibility of testing autologous monocytes for application of therapeutic genes in human CNS disease. Moreover, these data support the results from bone marrow grafts that circulating CD11b+ cells can enter the CNS without requiring the use of lethal irradiation.

Panel of synaptic protein ELISAs for evaluating neurological phenotype.

The purpose of this study was to develop ELISAs for key neural proteins, three synaptic and one glial, that exist in different intracellular compartments, which would be used as a measure of synaptic phenotype. These assays would be valuable to neurologically phenotype transgenic mouse models of human disease and also human disease itself using minimal amounts of post-mortem tissue. We showed that supernatant from crude brain tissue homogenates extracted in RIPA buffer containing 0.1% SDS bind to synaptophysin, synaptosome-associated protein of 25 kDa (SNAP-25), post-synaptic density-95 (PSD-95), and glial fibrillary acidic protein (GFAP) antibody pairs with high affinity and selectivity. Overall, RIPA + 0.1% SDS were more efficient than RIPA + 2% SDS or a buffer containing only 1% Triton-X-100. Diluting the brain extracts resulted in dose-dependent binding to the antibody pairs for each neural protein, with EC50s that varied from 8.6 microg protein for PSD-95 to 0.23 microg for GFAP. The assays were used to measure synaptic marker protein levels at various times during mouse development and GFAP in a model of disease accompanied by neuroinflammation. Comparison of ELISAs with Western blots by measuring marker levels in brain extract from developing mice showed a greater relative difference in values derived from ELISA. These ELISAs should be valuable to phenotype the synapse in neurological disease and their rodent models.

Abnormal post-translational and extracellular processing of brevican in plaque-bearing mice over-expressing APPsw.

Aggregation of amyloid-beta (Abeta) in the forebrain of Alzheimer's disease (AD) subjects may disturb the molecular organization of the extracellular microenvironment that modulates neural and synaptic plasticity. Proteoglycans are major components of this extracellular environment. To test the hypothesis that Abeta, or another amyloid precursor protein (APP) dependent mechanism modifies the accumulation and/or turnover of extracellular proteoglycans, we examined whether the expression and processing of brevican, an abundant extracellular, chondroitin sulfate (CS)-bearing proteoglycan, were altered in brains of Abeta-depositing transgenic mice (APPsw - APP gene bearing the Swedish mutation) as a model of AD. The molecular size of CS chains attached to brevican was smaller in hippocampal tissue from APPsw mice bearing Abeta deposits compared to non-transgenic mice, likely because of changes in the CS chains. Also, the abundance of the major proteolytic fragment of brevican was markedly diminished in extracts from several telencephalic regions of APPsw mice compared to non-transgenic mice, yet these immunoreactive fragments appeared to accumulate adjacent to the plaque edge. These results suggest that Abeta or APP exert inhibitory effects on proteolytic cleavage mechanisms responsible for synthesis and turnover of proteoglycans. As proteoglycans stabilize synaptic structure and inhibit molecular plasticity, defective brevican processing observed in Abeta-bearing mice and potentially end-stage human AD, may contribute to deficient neural plasticity.

Suppression of amyloid deposition leads to long-term reductions in Alzheimer's pathologies in Tg2576 mice.

In amyloid precursor protein (APP) models of amyloid deposition, the amount of amyloid deposits increase with mouse age. At a first approximation, the extent of amyloid accumulation may either reflect small excesses of production over clearance that accumulate over time or, alternatively, indicate a steady-state equilibrium at that age, reflecting the instantaneous excess of production over clearance, which increases as the organism ages. To discriminate between these options, we reversibly suppressed amyloid deposition in Tg2576 mice with the anti-Abeta antibody 2H6, starting at 8 months, just before the first histological deposits can be discerned. Six months later, we stopped the suppression and monitored the progression of amyloid accumulation in control APP mice and suppressed APP mice over the next 3 months. The accumulation hypothesis would predict that the rate of amyloid from 14 to 17 months would be similar in the suppressed and control mice, while the equilibrium hypothesis would predict that the increase would be faster in the suppressed group, possibly catching up completely with the control mice. The results strongly support the accumulation hypothesis, with no evidence of the suppressed mice catching up with the control mice as predicted by equilibrium models. If anything, there was a slower rate of increase in the suppressed APP mice than the control mice, suggesting that a slow seeding mechanism likely precedes a rapid fibrillogenesis in determining the extent of amyloid deposition.

Delayed administration of a matrix metalloproteinase inhibitor limits progressive brain injury after hypoxia-ischemia in the neonatal rat.

BACKGROUND: Hypoxia-ischemia (H-I) can produce widespread neurodegeneration and deep cerebral white matter injury in the neonate. Resident microglia and invading leukocytes promote lesion progression by releasing reactive oxygen species, proteases and other pro-inflammatory mediators. After injury, expression of the gelatin-degrading matrix metalloproteinases (MMPs), MMP-2 and MMP-9, are thought to result in the proteolysis of extracellular matrix (ECM), activation of cytokines/chemokines, and the loss of vascular integrity. Thus, therapies targeting ECM degradation and progressive neuroinflammation may be beneficial in reducing H-I - induced neuropathy. Minocycline has MMP-inhibitory properties and is both anti-inflammatory and neuroprotective. AG3340 (prinomastat) is an MMP inhibitor with high selectivity for the gelatinases. The purpose of this study was to determine whether these compounds could limit H-I--induced injury when administered at a delayed time point. METHODS: Sprague-Dawley rats were exposed to H-I at postnatal day 7 (P7), consisting of unilateral carotid artery ligation followed by 90 min exposure to 8% O2. Minocycline, AG3340, or vehicle were administered once daily for 6 days, beginning 24 hours after insult. Animals were sacrificed at P14 for neurohistological assessments. Immunohistochemistry was performed to determine the degree of reactive astrogliosis and immune cell activation/recruitment. Neural injury was detected using the Fluoro-Jade stain, a marker that identifies degenerating cells. RESULTS: CD11b and glial fibrillary acidic protein (GFAP) immunopositive cells increased in ipsilateral cortex after treatment with vehicle alone, demonstrating microglia/macrophage recruitment and reactive astrogliosis, respectively. Fluoro-Jade staining was markedly increased throughout the fronto-parietal cortex, striatum and hippocampus. Treatment with minocycline or AG3340 inhibited microglia/macrophage recruitment, attenuated astrogliosis and reduced Fluoro-Jade staining when compared to vehicle alone. CONCLUSION: The selective gelatinase inhibitor AG3340 showed equal efficacy in reducing neural injury and dampening neuroinflammation when compared to the anti-inflammatory compound minocycline. Thus, MMP-2 and MMP-9 may be viable therapeutic targets to treat neonatal brain injury.

Deglycosylated anti-Abeta antibody dose-response effects on pathology and memory in APP transgenic mice.

Anti-Abeta antibody administration to amyloid-depositing transgenic mice can reverse amyloid pathology and restore memory function. However, in old mice, these treatments also increase vascular leakage and promote formation of vascular amyloid deposits. Deglycosylated antibodies with reduced affinity for Fcgamma receptors and complement are associated with reduced vascular amyloid and microhemorrhage while retaining amyloid-clearing and memory-enhancing properties of native intact antibodies. In the current experiment, we investigated the effect of 3, 10, or 30 mg/kg of deglycosylated antibody (D-2H6) on amyloid pathology and cognitive behavior in old Tg2576 mice. We found that low doses of deglycosylated antibody appear more efficacious than higher doses in reducing pathology and memory loss in amyloid precursor protein (APP) transgenic mice. These data suggest that excess antibody unbound to antigen can interfere with antibody-mediated Abeta clearance, possibly by saturating the FcRn antibody transporter.

Adeno-associated viral (AAV) serotype 5 vector mediated gene delivery of endothelin-converting enzyme reduces Abeta deposits in APP + PS1 transgenic mice.

Reduction of Abeta deposition is a major therapeutic strategy in Alzheimer's disease (AD). The concentration of Abeta in the brain is modulated not only by Abeta production but also by its degradation. One of the proteases involved in the degradation of Abeta peptides is endothelin-converting enzyme (ECE). In this study, we investigated the effects of an intracranial administration of a seroptype 5 recombinant adeno-associated viral vector (rAAV) containing the ECE-1 synthetic gene on amyloid deposition in amyloid precursor protein (APP) plus presenilin-1 (PS1) transgenic mice. The rAAV vector was injected unilaterally into the right anterior cortex and hippocampus of 6-month-old mice, while control mice received an AAV vector expressing green fluorescent protein (GFP). Immunohistochemical testing for the hemagglutinin (HA) tag appended to ECE revealed strong expression in areas surrounding the injection sites but minimal expression in the contralateral regions. Immunohistochemical tests showed that Abeta decreases in the anterior cortex and hippocampus in mice receiving the ECE synthetic gene. Further, decreases in Congo red positive deposits were also observed in both regions. These results indicate that increasing the expression of beta-amyloid degrading enzymes through gene therapy is a promising approach to the treatment of AD.

Multimodal signaling by the ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) promotes neurite extension.

Aggregating proteoglycans (PG) bearing chondroitin sulfate (CS) side chains associate with hyaluronan and various secreted proteins to form a complex of extracellular matrix (ECM) that inhibits neural plasticity in the central nervous system (CNS). Chondroitinase treatment depletes PGs of their CS side chains and enhances neurite extension. Increasing evidence from in vivo models indicates that proteolytic cleavage of the PG core protein by members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of glutamyl-endopeptidases also promotes neural plasticity. The purpose of this study was to determine whether proteolytic action of the ADAMTSs influences neurite outgrowth in cultured neurons. Transfection of primary rat neurons with ADAMTS4 cDNA induced longer neurites, whether the neurons were grown on a monolayer of astrocytes that secrete inhibitory PGs or on laminin/poly-L-lysine substrate alone. Similar results were found when neurons were transfected with a construct encoding a proteolytically inactive, point mutant of ADAMTS4. Addition of recombinant ADAMTS4 or ADAMTS5 protein to immature neuronal cultures also enhanced neurite extension in a dose-dependent manner, an effect demonstrated to be dependent on the activation of MAP ERK1/2 kinase. These results suggest that ADAMTS4 enhances neurite outgrowth via a mechanism that does not require proteolysis but is dependent on activation of the MAP kinase cascade. Thus a model to illustrate multimodal ADAMTS activity would entail proteolysis of CS-bearing PGs to create a loosened matrix environment more favorable for neurite outgrowth, and enhanced neurite outgrowth directly stimulated by ADAMTS signaling at the cell surface.

Discordant localization of WFA reactivity and brevican/ADAMTS-derived fragment in rodent brain.

BACKGROUND: Proteoglycan (PG) in the extracellular matrix (ECM) of the central nervous system (CNS) may act as a barrier for neurite elongation in a growth tract, and regulate other characteristics collectively defined as structural neural plasticity. Proteolytic cleavage of PGs appears to alter the environment to one favoring plasticity and growth. Brevican belongs to the lectican family of aggregating, chondroitin sulfate (CS)-bearing PGs, and it modulates neurite outgrowth and synaptogenesis. Several ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) are glutamyl-endopeptidases that proteolytically cleave brevican. The purpose of this study was to localize regions of adult CNS that contain a proteolytic-derived fragment of brevican which bears the ADAMTS-cleaved neoepitope sequence. These regions were compared to areas of Wisteria floribunda agglutin (WFA) reactivity, a common reagent used to detect "perineuronal nets" (PNNs) of intact matrix and a marker which is thought to label regions of relative neural stability. RESULTS: WFA reactivity was found primarily as PNNs, whereas brevican and the ADAMTS-cleaved fragment of brevican were more broadly distributed in neuropil, and in particular regions localized to PNNs. One example is hippocampus where the ADAMTS-cleaved brevican fragment is found surrounding pyramidal neurons, in neuropil of stratum oriens/radiatum and the lacunosum moleculare. The fragment was less abundant in the molecular layer of the dentate gyrus. Mostly PNNs of scattered interneurons along the pyramidal layer were identified by WFA. In lateral thalamus, the reticular thalamic nucleus stained abundantly with WFA whereas ventral posterior nuclei were markedly immunopositive for ADAMTS-cleaved brevican. Using Western blotting techniques, no common species were reactive for brevican and WFA. CONCLUSION: In general, a marked discordance was observed in the regional localization between WFA and brevican or the ADAMTS-derived N-terminal fragment of brevican. Functionally, this difference may correspond to regions with varied prevalence for neural stability/plasticity.

Adeno-associated Viral (AAV) Serotype 5 Vector Mediated Gene Delivery of Endothelin-converting Enzyme Reduces Aß Deposits in APP + PS1 Transgenic Mice.

Reduction of Aß deposition is a major therapeutic strategy in Alzheimer's disease (AD). The concentration of Aß in the brain is modulated not only by Aß production but also by its degradation. One of the proteases involved in the degradation of Aß peptides is endothelin-converting enzyme (ECE). In this study, we investigated the effects of an intracranial administration of a seroptype 5 recombinant adeno-associated viral vector (rAAV) containing the ECE-1 synthetic gene on amyloid deposition in amyloid precursor protein (APP) plus presenilin-1 (PS1) transgenic mice. The rAAV vector was injected unilaterally into the right anterior cortex and hippocampus of 6-month-old mice, while control mice received an AAV vector expressing green fluorescent protein (GFP). Immunohistochemical testing for the hemagglutinin (HA) tag appended to ECE revealed strong expression in areas surrounding the injection sites but minimal expression in the contralateral regions. Immunohistochemical tests showed that Aß decreases in the anterior cortex and hippocampus in mice receiving the ECE synthetic gene. Further, decreases in Congo red positive deposits were also observed in both regions. These results indicate that increasing the expression of ß-amyloid degrading enzymes through gene therapy is a promising approach to the treatment of AD.

Versican and brevican are expressed with distinct pathology in neonatal hypoxic-ischemic injury.

The developing brain is uniquely susceptible to injury after exposure to hypoxia-ischemia (H-I). Lecticans are developmentally regulated in formative white matter and exert growth-inhibitory effects in several adult injury models, yet little is known regarding their role in neonatal H-I injury. The main objectives of this study were to examine the expression profiles of brevican and versican in rat using a standard H-I model and to determine whether altered expression was associated with distinct components of white and gray matter pathology. The H-I procedure in postnatal day 7 rats produced progressive injury limited to the ipsilateral hemisphere. Cresyl violet staining revealed severe cavitary infarctions at 14 and 21 days that were absent at 4 days. Cellular damage, as measured by glial fibrillary acidic protein and fractin immunoreactivity, occurred in cortical and subcortical gray matter at all end points. O4 sulfatide immunoreactivity was reduced in the external capsule, hippocampal fimbria, and corpus striatum at 4 days relative to that contralaterally, suggesting the loss of preoligodendrocytes. Brevican expression was reduced in the cortex and hippocampus at 4 days but was markedly elevated at later end points, localizing to regions of cellular damage both in and proximal to the lesion core. However, versican was reduced in the external capsule 4 days after H-I, a reduction that was sustained up to 21 days in white matter. These data demonstrate unique expression profiles for lecticans after neonatal H-I, suggesting brevican deposition is elevated in response to progressive gray matter injury, whereas diminished versican expression may be associated with deep cerebral white matter injury.

Pre-assembled clusters distort crystal nucleation kinetics in supersaturated lysozyme solutions.

Efficient determination of three-dimensional protein structures is critical for unraveling structure-function relationships and for supporting targeted drug design. A major impediment to these efforts is our lack of control over the nucleation and growth of high-quality protein crystals for X-ray structure determinations. While basic research on protein crystal growth mechanisms has provided valuable new insights, studies of crystal nucleation have been plagued by inconsistent and outright contradictory results. Using dynamic light scattering and SDS gel electrophoresis, we have investigated possible causes of these inconsistencies. We find that commercial sources of lyophilized hen-egg white lysozyme (HEWL) used in nucleation studies contain significant populations of large (approximately 100 nm), pre-assembled lysozyme clusters that can readily evade standard assays of sample purity. In supersaturated solutions, these clusters act as heterogeneous nucleation centers that enhance the rate of crystal nucleation and significantly deteriorate the quality of macroscopic crystals.

Intracranial administration of deglycosylated C-terminal-specific anti-Abeta antibody efficiently clears amyloid plaques without activating microglia in amyloid-depositing transgenic mice.

BACKGROUND: Antibodies against the Ass peptide clear Ass deposits when injected intracranially. Deglycosylated antibodies have reduced effector functions compared to their intact counterparts, potentially avoiding immune activation. METHODS: Deglycosylated or intact C-terminal specific high affinity anti-Abeta antibody (2H6) were intracranially injected into the right frontal cortex and hippocampus of amyloid precursor protein (APP) transgenic mice. The untreated left hemisphere was used to normalize for the extent of amyloid deposition present in each mouse. Control transgenic mice were injected with an antibody against a drosophila-specific protein (amnesiac). Tissues were examined for brain amyloid deposition and microglial responses 3 days after the injection. RESULTS: The deglycosylated 2H6 antibody had lower affinity for several murine Fcgamma receptors and human complement than intact 2H6 without a change in affinity for Ass. Immunohistochemistry for Abeta and thioflavine-S staining revealed that both diffuse and compact deposits were reduced by both antibodies. In animals treated with the intact 2H6 antibody, a significant increase in Fcgamma-receptor II/III immunostaining was observed compared to animals treated with the control IgG antibody. No increase in Fcgamma-receptor II/III was found with the deglycosylated 2H6 antibody. Immunostaining for the microglial activation marker CD45 demonstrated a similar trend. CONCLUSION: These findings suggest that the deglycosylated 2H6 is capable of removing both compact and diffuse plaques without activating microglia. Thus, antibodies with reduced effector functions may clear amyloid without concomitant immune activation when tested as immunotherapy for Alzheimer's disease.