The Role of Tissue Geometry in Spinal Cord Regeneration.

Unlike peripheral nerves, axonal regeneration is limited following injury to the spinal cord. While there may be reduced regenerative potential of injured neurons, the central nervous system (CNS) white matter environment appears to be more significant in limiting regrowth. Several factors may inhibit regeneration, and their neutralization can modestly enhance regrowth. However, most investigations have not considered the cytoarchitecture of spinal cord white matter. Several lines of investigation demonstrate that axonal regeneration is enhanced by maintaining, repairing, or reconstituting the parallel geometry of the spinal cord white matter. In this review, we focus on environmental factors that have been implicated as putative inhibitors of axonal regeneration and the evidence that their organization may be an important determinant in whether they inhibit or promote regeneration. Consideration of tissue geometry may be important for developing successful strategies to promote spinal cord regeneration.

Segregated neural explants exhibit co-oriented, asymmetric, neurite outgrowth.

Explants of embryonic chick sympathetic and sensory ganglia were found to exhibit asymmetric radial outgrowth of neurites under standard culture conditions with or without exogenous Nerve Growth Factor [NGF]. Opposing sides of an explant exhibited: a) differences in neurite length and, b) differences in neurite morphology. Strikingly, this asymmetry exhibited co-orientation among segregated, neighboring explants. The underlying mechanism(s) of the asymmetry and its co-orientation are not known but appear to depend on cell clustering because dissociated sympathetic neurons do not exhibit co-orientation whereas re-aggregated clusters of cells do. This emergent behavior may be similar to the community effect described in other cell types. If a similar phenomenon exists in the embryo, or in maturity, it may contribute to the establishment of proper orientation of neurite outgrowth during development and/or injury-induced neuronal plasticity.

Reduced sympathetic neurite outgrowth on uterine tissue sections from rats treated with estrogen.

In order to evaluate the contribution of substrate-bound factors to the extent and patterning of the sympathetic innervation of rat uterus following estrogen treatment, superior cervical ganglion explants from neonatal and adult ovariectomized rats were cultured on tissue sections of fresh frozen uterus from adult ovariectomized rats treated with estrogen or a vehicle. The main findings were: (1) neurite growth was greatly influenced by histological features of the underlying section; (2) on myometrial sections, neurites followed the orientation of the main axis of the longitudinally sectioned muscle cells; (3) neurites showed limited growth on transversally sectioned smooth muscle; (4) neuritic patterning was unaffected by a reduction in migrating ganglionic non-neuronal cells; (5) neurite outgrowth, but not non-neural cell migration, was markedly reduced on myometrial sections from rats treated with estrogen. These results suggest that adult myometrium continues to provide signals allowing the organotypic patterning and growth of sympathetic axons, that estrogen treatment modifies myometrial substrate properties so that it is less supportive for sympathetic neurite growth, and that adult sympathetic neurons retain their ability to recognize substrate-bound cues present in the myometrium. On endometrial sections, neurites formed radially symmetric halos, which were reduced in size on estrogen-treated endometrial substrates. Thus, changes in the neuritogenic capacity of the uterus underlie plasticity in uterine sympathetic nerves, and alterations in substrate-bound factors contribute to the diminished receptivity of the estrogenized uterus to its sympathetic innervation.

Meta-analysis of APOE4 allele and outcome after traumatic brain injury.

There is conflicting evidence regarding a possible association between the apolipoprotein E4 (APOE4) allele and the consequences of traumatic brain injury (TBI). Our aim was to carry out a meta-analysis of cohort studies of sufficient rigor to determine whether the presence of the APOE4 allele contributes to initial injury severity and/or poor outcome following TBI. MEDLINE, EMBase, CBMdisc, and CNKI databases were searched for literature published from January 1993 to October 2007. Of the 100 identified studies, 14 cohort studies were selected for analysis based on comprehensive quality assessment using a standardized scale. Data from the 14 eligible cohort studies included a total of 2527 participants, 736 with and 1791 without the APOE4 allele. The APOE4 allele was not associated with initial injury severity of TBI. The pooled RR were 1.11 (95% confidence interval [CI], 0.91 to 1.35) for severe injury, 1.06 (95% CI, 0.86-1.31) for moderate injury and 0.93 (95% CI, 0.81-1.06) for mild injury. However, the APOE4 allele was significantly associated with a poor outcome of TBI at 6 months after injury (RR = 1.36; 95% CI, 1.04-1.78). The association remained significant in sensitivity tests. This meta-analysis indicates that the presence of the APOE4 allele is not associated with the initial severity of brain injury following TBI but is associated with increased risk of poor long-term outcome at 6 months after injury.

A simple, efficient tool for assessment of mice after unilateral cortex injury.

A refined battery of neurological tests, SNAP (Simple Neuroassessment of Asymmetric Impairment), was developed and validated to efficiently assess neurological deficits induced in a mouse model of traumatic brain injury. Four to 7-month old mice were subjected to unilateral controlled cortical impact or sham injury (craniectomy only). Several behavioral tests (SNAP, beam walk, foot fault, and water maze) were used to assess functional deficits. SNAP was unique among these in that it required no expensive equipment and was performed in less than 5 min per mouse. SNAP demonstrated a high level of sensitivity and specificity as determined by receiver-operator characteristics curve analysis. Interrater reliability was good, as determined by Cohen's Kappa method and by comparing the sensitivity and specificity across various raters. SNAP detected deficits in proprioception, visual fields, and motor strength in brain-injured mice at 3 days, and was sensitive enough to detect magnitude and recovery of injury. The contribution of individual battery components changed as a function of time after injury, however, each was important to the overall SNAP score. SNAP provided a sensitive, reliable, time-efficient and cost-effective means of assessing neurological deficits in mice after unilateral brain injury.

ProNGF, sortilin, and age-related neurodegeneration.

Several studies have sought to demonstrate that neurodegeneration during disease and in old age is associated with reduced neurotrophic support. Little positive evidence has been forthcoming, either in relation to the availability of neurotrophins or to expression and function of the relevant receptors. Recently, a novel way in which neurotrophins could contribute to neurodegeneration has been suggested. In contrast to the well-known neurotrophic functions of the mature beta-form of nerve growth factor (mNGF), its precursor proNGF has recently been shown to be abundant in the adult brain and in the brains of patients with Alzheimer's disease. proNGF is synthesized as 25 and 32 kDa isoforms, which are glycosylated to form a principal 40 kDa species. Studies of the cortical targets of NGF-responsive basal forebrain neurons show that the 40 kDa form of proNGF is secreted in response to nerve stimulation, along with the proteases needed to generate the 13 kDa mNGF, or to degrade it. We have recently found that levels of 40 kDa proNGF are elevated in the aging brain and also in targets of peripheral NGF-responsive neurons. proNGF has been shown to be neurotoxic when bound in a heterotrimer with the p75 receptor and the receptor sortilin (identical to the neurotensin receptor NTS3). Interestingly, we find that sortilin levels increase in aged central and peripheral neurons, perhaps making these neurons more vulnerable to age-related increases in proNGF. Whether elevated levels of proNGF in targets or of sortilin in neurons contribute to known patterns of age- and disease-related neurodegeneration has not been previously investigated. Using in vitro models, our preliminary data now indicate that proNGF is indeed neurotoxic for aged, but not young, NGF-responsive basal forebrain and sympathetic neurons and that blockade of sortilin rescues proNGF-induced cell death. We therefore propose that increased proNGF in targets combined with increased sortilin expression in projecting neurons contributes to age-related neuronal atrophy and degeneration.

Global expression of NGF promotes sympathetic axonal growth in CNS white matter but does not alter its parallel orientation.

Axonal regeneration is normally limited after injuries to CNS white matter. Infusion of neurotrophins has been successful in promoting regenerative growth through injured white matter but this growth generally fails to extend beyond the infusion site. These observations are consistent with a chemotropic effect of these factors on axonal growth and support the prevailing view that neurotrophin-induced axonal regeneration requires the use of gradients, i.e., gradually increasing neurotrophin levels along the target fiber tract. To examine the potential of global overexpression of neurotrophins to promote, and/or modify the orientation of, regenerative axonal growth within white matter, we grafted nerve growth factor (NGF) responsive neurons into the corpus callosum of transgenic mice overexpressing NGF throughout the CNS under control of the promoter for glial fibrillary acidic protein. One week later, glial fibrillary acidic protein and chondroitin sulfate proteoglycan immunoreactivity increased within injured white matter around the grafts. NGF levels were significantly higher in the brains of transgenic compared with non-transgenic mice and further elevated within injury sites compared with the homotypic region of the non-injured side. Although there was minimal outgrowth from neurons grafted into non-transgenic mice, extensive parallel axonal regeneration had occurred within the corpus callosum up to 1.5 mm beyond the astrogliotic scar (the site of maximum NGF expression) in transgenic mice. These results demonstrate that global overexpression of neurotrophins does not override the constraints limiting regenerative growth to parallel orientations and suggest that such factors need not be presented as positive gradients to promote axonal regeneration within white matter.

Null mutations for exon III and exon IV of the p75 neurotrophin receptor gene enhance sympathetic sprouting in response to elevated levels of nerve growth factor in transgenic mice.

Under normal conditions, expression of the p75 neurotrophin receptor (p75NTR) by sympathetic neurons can increase the affinity of the signaling receptor, trkA, to target-derived nerve growth factor (NGF) at distal axons. We have previously reported that sprouting of sympathetic axons into NGF-rich target tissues is enhanced when p75NTR expression is perturbed, leading to the postulate that p75NTR may restrain sympathetic sprouting in response to elevated NGF levels. These observations were made using mice having a null mutation of the third p75NTR exon, a line that may express a hypomorphic form of this receptor. Since mice carrying a null mutation of the fourth p75NTR exon may not express a similar splice variant, we sought to determine whether these animals possess the same phenotype of enhanced sympathetic sprouting in response to elevated levels of NGF. Both lines of transgenic mice lacking p75NTR displayed similar degrees of sympathetic axonal sprouting into the cerebellum and trigeminal ganglia, two target tissues having elevated levels of NGF protein. Furthermore, the densities of sympathetic axons in both targets were significantly greater than those observed in age-matched NGF transgenic siblings expressing full-length p75NTR. Our new findings provide a comparative analysis of the phenotype in two independent mutations of the same neurotrophin receptor, revealing that p75NTR plays an important role in restricting sympathetic sprouting in response to higher NGF levels.

The receptor-binding region of human apolipoprotein E has direct anti-infective activity.

BACKGROUND: The APOE genotype has a uniquely strong influence on the outcome of viral infection. The mechanism is unknown, although one possibility is direct inhibition of viral entry into cells. METHODS: We have examined the direct anti-infective activity of a peptide analogue of the receptor-binding region of apolipoprotein E (apoE) that is known as "apoE dimer tandem repeat peptide" (apoEdp) and has previously been shown to mimic some of the biological effects of apoE and that recently was shown to bind low-density lipoprotein receptor-related protein. RESULTS: apoEdp has activity against herpes simplex virus types 1 and 2, human immunodeficiency virus, Pseudomonas aeruginosa, and Staphylococcus aureus; concentrations in the range of 1-20 micromol/L inhibit infection by 50%. These biological actions depend on adoption of an alpha -helical structure, as has been found for other biological effects of apoE peptides. The peptide interferes with the earliest stages of viral infection, preventing viral attachment and exerting a mild virucidal action. In addition, an N-terminal fragment of apoE that also contains this binding domain has antiviral activity. CONCLUSIONS: These data suggest that human apoE or fragments containing the receptor-binding domain may contribute to innate immunity to viral infection by direct disruption of viral particles and/or inhibition of viral attachment, thus reducing viral entry.

'Mature' nerve growth factor is a minor species in most peripheral tissues.

The classic neurotrophin hypothesis is based on the idea that innervating neurons derive 'mature' neurotrophin provided by the target for their survival. Yet large precursor forms of the neurotrophin nerve growth factor (NGF) have been reported in both central and peripheral tissues. In the present study, immunoblotting was used to survey peripheral tissues containing NGF-responsive neurons and to characterize various NGF species. These results demonstrate that 'mature' forms of NGF, i.e., the 13 and 16kDa species, are rare in sympathetic and sensory ganglia and in their peripheral targets, and that large molecular weight NGF precursors are abundant. In addition, certain NGF forms predominate in a given tissue, with each tissue exhibiting a characteristic NGF expression pattern. These findings suggest that NGF processing in peripheral tissues and in NGF-responsive ganglia may involve a variety of NGF species.

Progress toward identification of protease activity involved in proteolysis of apolipoprotein e in human brain.

Apolipoprotein E (apoE) genotype is the single most important genetic risk factor for the most common (sporadic) form of Alzheimer's disease (AD). Increasing evidence supports the hypothesis that the presence of the E4 isoform of this cholesterol-binding protein contributes directly to disease risk, age of onset, and severity of the neuropathology. For example, studies in transgenic mice demonstrate that apoE is necessary for the formation of plaques with neuritic pathology. The precise mechanism by which apoE contributes to the disease remains unknown. However, several lines of investigation from a number of laboratories now point to a role for proteolytic fragments of apoE in the formation of both plaques and tangles, the two pathological hallmarks of the disease. In particular, the C-terminal portion of apoE has been implicated in binding to amyloid and is localized to plaques. The N-terminal domain, on the other hand, is neurotoxic in culture and has been localized to, and implicated in the formation of, neurofibrillary tangles. These results suggest that inhibition of apoE proteolysis is a potential therapeutic strategy for AD. Using human brain homogenates, we have determined that proteolysis of apoE is greatest at acidic pH and can be inhibited by compounds targeting aspartic proteases. The feasibility of screening candidate inhibitors is supported by both ELISA and immunoblotting methods. Future studies will use a combination of in vitro and in vivo assays to test the efficacy of the most effective compounds for their ability to inhibit apoE proteolysis in human brain and apoE transgenic mouse brain tissue.

Apolipoprotein E is a prime suspect, not just an accomplice, in Alzheimer's disease.

There is now a large body of evidence suggesting that apolipoprotein E (apoE) genotype is the single most important genetic risk factor for the most common (sporadic) form of Alzheimer's disease. Yet in proportion to the total number of investigations in this field, relatively few groups are studying the contribution of this cholesterol-binding protein to disease risk and severity. Of those that are, a major focus is on the impact of apoE on amyloid-related mechanisms of disease. I argue here that apoE should be considered a major culprit in its own right, not simply in a supporting role. The argument is based on several lines of evidence, including the fact that apoE is associated with both plaques and tangles, the overwhelming evidence for genetic risk of the disease attributed to apoE, increasing evidence that apoE might also modify risk of other nonamyloidogenic neurological diseases, neurotoxicity attributed to apoE and/or proteolytic fragments of apoE, negative consequences of transgenic expression of apoE4 in mice, and genetic evidence for polymorphisms that increase both apoE expression and disease risk, regardless of isoform.

Apolipoprotein E-related neurotoxicity as a therapeutic target for Alzheimer's disease.

Apolipoprotein E (apoE) remains the most important genetic risk factor for the development of Alzheimer's disease (AD). Still elusive, the role of apoE is under intense investigation. We propose that proteolysis of apoE in the brain leads to two major fragments, N- and C-terminal apoE, each of which would drive a different neuropathological pathway. N-terminal fragments of apoE are implicated in neurotoxicity, and C-terminal fragments might play a role in amyloid deposition and plaque formation. The greater risk of AD associated with the E4 isoform might relate to its greater neurotoxicity. Drugs that either directly inhibit the toxic effects of apoE or prevent the production of apoE fragments may provide novel therapeutic approaches to the treatment of AD and other disorders in which apoE is implicated.

Inhibition of apolipoprotein E-related neurotoxicity by glycosaminoglycans and their oligosaccharides.

Apolipoprotein E (apoE) has been genetically linked to late-onset Alzheimer's disease (AD). The role of this lipid-transport protein in AD remains to be established. One hypothesis is that apoE, particularly the apoE4 isoform, may have neurotoxic effects as demonstrated using apoE-related synthetic peptides and the N-terminal fragment of apoE. ApoE is a heparan-sulfate binding protein, and apoE peptide neurotoxicity can be blocked by heparin and prevented by degrading heparan sulfate or inhibiting its biosynthesis. The possibility that heparin inhibition of toxicity is mediated by a specific oligosaccharide sequence was investigated using a bioassay to determine the inhibition of apoE peptide toxicity by glycosaminoglycans and purified glycosaminoglycan oligosaccharides. Studies on modified heparins showed that the presence of N-sulfo groups and either 2- or 6-O sulfo groups were required for inhibition of toxicity. Heparin oligosaccharides with eight or more saccharide residues with seven O-sulfo groups and four N-sulfo groups exhibited potent inhibition. Larger oligosaccharides, and heparin and heparan sulfate polymers, afforded comparable, or somewhat better, protective effects but also caused clumping and detachment of cells when administrated alone.