PDIA3 inhibits mitochondrial respiratory function in brain endothelial cells and C. elegans through STAT3 signaling and decreases survival after OGD.

BACKGROUND: Protein disulfide isomerase A3 (PDIA3, also named GRP58, ER-60, ERp57) is conserved across species and mediates protein folding in the endoplasmic reticulum. PDIA3 is, reportedly, a chaperone for STAT3. However, the role of PDIA3 in regulating mitochondrial bioenergetics and STAT3 phosphorylation at serine 727 (S727) has not been described. METHODS: Mitochondrial respiration was compared in immortalized human cerebral microvascular cells (CMEC) wild type or null for PDIA3 and in whole organism C. Elegans WT or null for pdi-3 (worm homologue). Mitochondrial morphology and cell signaling pathways in PDIA3-/- and WT cells were assessed. PDIA3-/- cells were subjected to oxygen-glucose deprivation (OGD) to determine the effects of PDIA3 on cell survival after injury. RESULTS: We show that PDIA3 gene deletion using CRISPR-Cas9 in cultured CMECs leads to an increase in mitochondrial bioenergetic function. In C. elegans, gene deletion or RNAi knockdown of pdi-3 also increased respiratory rates, confirming a conserved role for this gene in regulating mitochondrial bioenergetics. The PDIA3-/- bioenergetic phenotype was reversed by overexpression of WT PDIA3 in cultured PDIA3-/- CMECs. PDIA3-/- and siRNA knockdown caused an increase in phosphorylation of the S727 residue of STAT3, which is known to promote mitochondrial bioenergetic function. Increased respiration in PDIA3-/- CMECs was reversed by a STAT3 inhibitor. In PDIA3-/- CMECs, mitochondrial membrane potential and reactive oxygen species production, but not mitochondrial mass, was increased, suggesting an increased mitochondrial bioenergetic capacity. Finally, PDIA3-/- CMECs were more resistant to oxygen-glucose deprivation, while STAT3 inhibition reduced the protective effect. CONCLUSIONS: We have discovered a novel role for PDIA3 in suppressing mitochondrial bioenergetic function by inhibiting STAT3 S727 phosphorylation.

Vitamin-D receptor agonist calcitriol reduces calcification in vitro through selective upregulation of SLC20A2 but not SLC20A1 or XPR1.

Vitamin D deficiency (hypovitaminosis D) causes osteomalacia and poor long bone mineralization. In apparent contrast, hypovitaminosis D has been reported in patients with primary brain calcifications ("Fahr's disease"). We evaluated the expression of two phosphate transporters which we have found to be associated with primary brain calcification (SLC20A2, whose promoter has a predicted vitamin D receptor binding site, and XPR1), and one unassociated (SLC20A1), in an in vitro model of calcification. Expression of all three genes was significantly decreased in calcifying human bone osteosarcoma (SaOs-2) cells. Further, we confirmed that vitamin D (calcitriol) reduced calcification as measured by Alizarin Red staining. Cells incubated with calcitriol under calcifying conditions specifically maintained expression of the phosphate transporter SLC20A2 at higher levels relative to controls, by RT-qPCR. Neither SLC20A1 nor XPR1 were affected by calcitriol treatment and remained suppressed. Critically, knockdown of SLC20A2 gene and protein with CRISPR technology in SaOs2 cells significantly ablated vitamin D mediated inhibition of calcification. This study elucidates the mechanistic importance of SLC20A2 in suppressing the calcification process. It also suggests that vitamin D might be used to regulate SLC20A2 gene expression, as well as reduce brain calcification which occurs in Fahr's disease and normal aging.

Prolonged local neurotrophin-3 infusion reduces ipsilateral collateral sprouting of spared corticospinal axons in adult rats.

The corticospinal tract is widely used to study regeneration and is essential for voluntary movements in humans. In young rats, corticospinal axons on the uninjured side sprout and grow into the denervated side. Neurotrophin-3 (NT-3) induces such crossed collateral sprouting in adults. We investigated whether local intraspinal NT-3 infusions would promote collateral sprouting of spared corticospinal terminals from within a partially denervated side, as this would be more appropriate for enhancing function of unilateral and specific movements. Adult rats received a partial bilateral transection of the pyramids, leaving approximately 40% of each tract intact. Vehicle or vehicle plus NT-3 (3 or 10 microg/day) was infused for 14 days into the left side of the cervical (C5/6) or lumbar (L2) cord. The corticospinal processes on the left side were anterogradely traced with cholera toxin B (CTB; which labeled gray matter processes more robustly than biotinylated dextran amine) injected into the front or hind limb area of the right sensorimotor cortex, respectively, 3 days before analysis. Unexpectedly, approximately 40% fewer CTB-labeled corticospinal processes were detectable in the cervical or lumbar gray matter of NT-3-treated rats than in vehicle-infused ones. Vehicle-infused injured rats had more corticospinal processes in the center of the cord than normal rats, evidence for lesion-induced collateral sprouting. NT-3 caused sprouting of local calcitonin gene-related peptide-positive fibers. These results suggest that NT-3 reduces collateral sprouting of spared corticospinal axons from within the denervated regions, possibly because of the injury environment or by increasing sprouting of local afferents. They identify an unexpected context-dependent outgrowth inhibitory effect of NT-3.

alpha6beta1 integrin directs migration of neuronal precursors in adult mouse forebrain.

New neuroblasts are constantly generated in the adult mammalian subventricular zone (SVZ) and migrate via the very-restricted rostral migratory stream (RMS) to the olfactory bulb, where they differentiate into functional neurons. Several facilitating and repulsive molecules for this migration have been identified, but little is known about chemoattractive molecules involved in the directed nature of this migration in vivo. Here, we investigated the role of the alpha6beta1 integrin, and its ligand, laminin, in controlling guidance of the migrating neuroblasts in adult mice. Immunostaining for the alpha6beta1 integrin was present in neuroblasts and their processes in the anterior/rostral SVZ and the RMS. Inhibition of the endogenous alpha6 or beta1 subunit with locally injected antibodies disrupted the cohesive nature of the RMS, but did not kill the neuroblasts. Infusion of a 15 a.a. peptide, representing the E8 domain of the laminin alpha chains that bind alpha6beta1 integrin, into the neostriatum redirected the neuroblasts away from the RMS towards the site of infusion. Injection of a narrow tract of intact laminin also drew the neuroblasts away from the RMS, but in a more restricted localization. These results suggest a critical role for integrins and laminins in adult SVZ-derived neuroblast migration. They also suggest that integrin-based strategies could be used to direct or restrict neuroblasts to CNS regions where they are needed for cell replacement therapies in the nervous system.

Endogenous and exogenous ciliary neurotrophic factor enhances forebrain neurogenesis in adult mice.

Neurogenesis in the adult mammalian CNS occurs in the subventricular zone (SVZ) and dentate gyrus. The receptor for ciliary neurotrophic factor (CNTF), CNTFRalpha, is expressed in the adult subventricular zone. Because the in vitro effects of CNTF on neural precursors have been varied, including proliferation and differentiation into neurons or glia, we investigated its role in vivo. Injection of CNTF in the adult C57BL/6 mice forebrain increased the number of cells labeled with ip BrdU in both neurogenic regions. In the dentate gyrus, CNTF also appeared to enhance differentiation of precursors into neurons, i.e., increased the proportion of NeuN+/BrdU+ cells from approximately 14 to approximately 29%, but did not affect differentiation into astrocytes (GFAP+) or oligodendrocytes (CNPase+). In the SVZ, CNTF increased the proportion of GFAP+/BrdU+ cells from approximately 1 to approximately 2%. CNTF enhanced the distance of migration of new neurons into the granule cell layer. Intraventricular injection of neutralizing anti-CNTF antibodies reduced the number of BrdU-labeled cells in the SVZ. These results suggest that endogenous CNTF regulates adult neurogenesis by increasing proliferation of neural stem cells and/or precursors. Alternatively, CNTF could maintain cells longer in the S-phase, resulting in increased BrdU labeling. In the neurogenic region of the SVZ, CNTFRalpha was exclusively present in GFAP-positive process-bearing cells, suggesting that CNTF affects neurogenesis indirectly via neighboring astroglia. Alternatively, these cells may be part of the neural precursor lineage. The restricted expression of CNTF within the nervous system makes it a potential selective drug target for cell replacement strategies.

Marked differences in olfactory sensitivity and apparent speed of forebrain neuroblast migration in three inbred strains of mice.

In the adult forebrain, new neuroblasts constantly migrate from the subventricular zone along the rostral migratory stream to the olfactory bulb, where many become neurons. It is unclear whether this process is different in commonly used mouse strains and whether it is related to olfactory function. Adult male BALB/c, C57BL/6, and 129/S1 (formerly 129SV) mice were tested for olfactory sensitivity plus discrimination, using male mouse urine from the two other strains. BALB/c mice had the greatest olfactory sensitivity, followed by 129/S1, and C57BL/6 mice, by an order of magnitude each. Newly formed cells were pulse-labeled for 3 h with i.p. 5-bromo-2'-deoxyuridine (BrdU) injections and the animals analyzed 24 h later. In 129/S1 mice, a greater proportion of neuroblasts were present closer to the olfactory bulb than in BALB/c mice, followed by C57BL/6 mice. The total number of BrdU-labeled cells did not differ, suggesting differences in migration and not proliferation. The impaired olfactory function in C57BL/6 mice might be caused by the reduced number of neuroblasts that reach the olfactory bulbs. However, olfactory function in BALB/c and 129/S1 mice did not correlate with their putative migration speed, suggesting a more complex nature of cellular processes that contribute to olfactory function. These results caution against comparing studies of olfactory function or neural precursors that use different strains of mice, and question the use of C57BL/6 mice as a "normal" strain or as transgenic background. Perhaps more importantly, the results point to an opportunity to identify genes that regulate olfactory function and neuroblast behavior.

Tyrosine phosphatase inhibition enhances neurotrophin potency and rescues nigrostriatal neurons in adult rats.

Neurotrophic factors regulate a variety of cellular processes, including neuronal survival during development and after injury. For instance, brain-derived neurotrophic factor (BDNF) can prevent the death of dopaminergic substantia nigra neurons in rats. Most neurotrophic factor receptors, such as TrkB for BDNF, are tyrosine kinases whose signaling is terminated by protein tyrosine phosphatases (PTPs). We tested the idea that inhibition of PTPs, and thus potentially enhancement of the efficiency of endogenous trophic factors and their receptors, would lead to increased neuronal survival. After a 2-week infusion of the small PTP inhibitor molecule peroxovanadium (pVa, pervanadate) close to the substantia nigra of adult rats, up to 66% of axotomized substantia nigra neurons had survived, compared to only 33% in control rats infused with PBS. PVa most likely affected TrkB and/or downstream signaling molecules, as ineffective doses of BDNF and pVa had a synergistic effect when given simultaneously, rescuing 82% of the neurons. PVa stimulated tyrosine hydroxylase (TH) expression in the noninjured substantia nigra but did not prevent axotomy-induced loss of TH. These results raise the possibility that PTP inhibition can prevent neuronal death by enhancing neurotrophic factor signaling pathways in the adult mammalian nervous system, identifies an important role for PTPs in neuronal functioning, and points to a novel small molecule treatment approach for neurologic disorders

Delayed NGF infusion fails to reverse axotomy-induced degeneration of basal forebrain cholinergic neurons in adult p75(LNTR)-deficient mice.

The p75 low-affinity neurotrophin receptor (p75(LNTR)) appears to have various functions that include enhancing nerve growth factor (NGF)-mediated survival by increasing TrkA (high-affinity NGF receptor) efficiency, and mediating apoptosis by acting as a ligand-regulated pro-apoptotic receptor. Here, we investigated the role of p75(LNTR) for adult cholinergic basal forebrain neurons by comparing neuronal responses to injury in control and p75(LNTR)-deficient mice. In both types of mice, approximately 70% of the cholinergic neurons in the ipsilateral medial septum had lost their markers choline acetyltransferase and tyrosine kinase A by 28 days following unilateral transection of the dorsal septohippocampal pathway (fimbria fornix). A 7-day delayed infusion of NGF that started 28 days after the injury resulted in reversal of choline acetyltransferase expression and cell atrophy in control, but not in p75(LNTR)-deficient, mice. This lack of response to delayed NGF treatment in p75(LNTR)-deficient mice was most likely not due to cell death, as all of the septohippocampal neurons, labeled with Fluorogold before the lesion, were present at 28 days post-lesion, similar to control mice. p75(LNTR)-deficient cholinergic neurons can respond to NGF as they were protected by NGF infusions that started immediately after the injury. These observations, the fact that lesioned p75(LNTR)-deficient neurons atrophy faster, and that non-lesioned neurons hypertrophy in response to NGF in control but not in p75(LNTR)-deficient mice, suggest that p75(LNTR) is needed for tyrosine kinase A and NGF signaling efficiency.In conclusion, during adulthood p75(LNTR) appears to play a beneficial role in the response of cholinergic neurons to injury, consistent with the proposed role of p75(LNTR) in the enhancement of TrkA signaling and the transport of neurotrophins by these neurons.

Retrograde tracing techniques influence reported death rates of adult rat nigrostriatal neurons.

Injury often causes loss of neuronal markers and prior retrograde labeling can circumvent this problem of identification. We have previously used a time-consuming protocol for labeling all dopaminergic substantia nigra pars compacta neurons in adult rats by injecting the fluorescent tracer DiI into six sites throughout each neostriatum. Here, 2 weeks after injection of DiI into two central locations, only half of these nigrostriatal neurons were labeled. With six sites, more medial and lateral neurons were labeled, and also more in the midportion along the medial-lateral extent of the pars compacta. Less than 0.5% of the contralateral neurons were labeled. Two injections of Fluorogold also labeled fewer neurons, but their morphology was clearer. Two to 4 weeks after injection of the neurotoxin 6-OHDA into the two neostriatal sites, the total number of surviving neurons appeared greater with six sites of DiI than with two. However, within the middle region of the nigra, survival was lower with the six sites. This suggests that neurons that project outside the two central striatal tracer and 6-OHDA injection regions may be spared initially, but that those in the midportion that project to the central region are more vulnerable with the six-site protocol. Some reports suggest that Fluorogold prelabeling increases neuronal death. Here, survival after 6-OHDA or axotomy was similar with DiI or Fluorogold. These results suggest that because of a complex projection pattern of the nigrostriatal neurons, detailed quantification of neuronal survival should rely on extensive labeling. However, for drug screening purposes, faster labeling with Fluorogold using two sites is more suitable and should provide reliable data.

Neutralizing antibodies against neurite growth inhibitor NI-35/250 do not promote regeneration of sensory axons in the adult rat spinal cord.

Neutralization of the myelin-associated neurite growth inhibitors NI-35 and NI-250 by IN-1 antibodies can promote axonal regeneration of several types of central nervous neurons. Here, we investigated in adult rats whether IN-1 can promote regeneration of ascending sensory axons across a peripheral nerve bridge back into the spinal cord. IN-1 was administered by hybridoma cells injected in the cerebral cortex or thoracic cord, its presence confirmed in tissue sections and cerebrospinal fluid, and its effectiveness demonstrated in co-cultures of oligodendrocytes and sensory neurons. With a two week infusion of control vehicle into the dorsal spinal cord 3 mm rostral to the nerve graft, only 3+/-2% of the anterogradely labeled sensory fibers present at the rostral end of the nerve graft had grown up to 0.5 mm, but not farther into the spinal cord. A similar limited extent of regeneration was seen with IN-1 or with infusion of Dantrolene, an inhibitor of NI-35/250 activity in vitro. With infusion of nerve growth factor rostral to the nerve graft, 40% of the fibers at the rostral end of the graft were found at 0.5 mm, 34% at 1 mm, 24% at 2 mm and 14% at 3 mm (the infusion site) into the spinal cord. Treatment with IN-l antibodies did not enhance the growth-promoting effects of nerve growth factor. We suggest that the neurite growth inhibitors NI-35 or NI-250 do not play a major inhibitory role in the regeneration of the ascending sensory fibers across a nerve bridge and back into the spinal cord of the adult rat.

SEK1/MKK4, c-Jun and NFKappaB are differentially activated in forebrain neurons during postnatal development and injury in both control and p75NGFR-deficient mice.

The common neurotrophin receptor (p75NGFR) can signal in vitro through activation of the c-Jun N-terminal kinase (JNK) pathway and nuclear translocation of NFKappaB. Activation of JNK and its substrate c-Jun can lead to apoptosis. We investigated these activities in vivo by comparing immunoreactivity for phosphorylated(p) SEK-1 (or MKK4, which activates JNK), c-Jun (ser63, ser73) and nuclear translocation of NFKappaB-p50 in tissue sections through the forebrain of control and p75NGFR-deficient mice. During postnatal development, SEK1p-immunoreactivity was detectable in p75NGFR-positive cholinergic neurons and p75NGFR-negative neurons throughout the forebrain in control mice. During development, few cells contained c-Junp, although many neurons contained c-Jun. No obvious c-Jun immunostaining was present in the adult forebrain. At any age, NFKappaB-p50 immunoreactivity was seen in nuclei of most cells throughout the forebrain. Following fimbria fornix transection in adult mice, few basal forebrain neurons contained SEK1p while many axotomized choline acetyltransferase (ChAT)-positive neurons contained c-Junp and nuclear NFKappaB-p50. The immunostaining patterns of SEK1p, c-Junp and NFKB during development and following injury were largely similar in p75NGFR-deficient mice. During development, cells throughout the forebrain had TdT-mediated dUTP-biotin nick end labelling (TUNEL)-labelling (a potential marker for apoptosis), however, their presence was not predicted by number of neurons stained for SEK1p or c-Junp. These results suggest that the expected activation of the JNK pathway by p75NGFR, as well as the expected relationship between SEK1 and downstream activation of c-Jun do not occur in the mammalian forebrain. Also, these results suggest that this activation does not necessarily lead to cell death.

Cholinergic medial septum neurons do not degenerate in aged 129/Sv control or p75(NGFR)-/-mice.

Cholinergic medial septum neurons express TrkA and p75 nerve growth factor receptor (p75(NGFR)) and interactions between TrkA and p75(NGFR) are necessary for high-affinity binding and signaling of nerve growth factor (NGF) through TrkA. In adult p75(NGFR)-deficient (-/-) mice, retrograde transport of NGF and other neurotrophins by these neurons is greatly reduced, however, these neurons maintain their cholinergic phenotype and size. Reduced transport of NGF has been proposed to play a role in Alzheimer's disease. Here, we investigated whether chronic and long-term absence of p75(NGFR) (and possibly reduced NGF transport and TrkA binding) would affect the cholinergic septohippocampal system during aging in mice. In young (6-8 months), middle aged (12-18 months), and aged (19-23 months) 129/Sv control mice the total number of choline acetyltransferase-positive medial septum neurons and the mean diameter and cross sectional area of the cholinergic cell bodies were similar. The cholinergic hippocampal innervation, as measured by the density of acetylcholinesterase-positive fibers in the outer molecular layer of the dentate gyrus was also similar across all ages. These parameters also did not change during aging in p75(NGFR) -/- mice and the number and size of the choline acetyltransferase-positive neurons and the cholinergic innervation density were largely similar as in control mice at all ages. These results suggest that p75(NGFR) does not play a major role in the maintenance of the number or morphology of the cholinergic basal forebrain neurons during aging of these mice. Alternatively, p75(NGFR) -/- mice may have developed compensatory mechanisms in response to the absence of p75(NGFR).

Naloxone prevents microglia-induced degeneration of dopaminergic substantia nigra neurons in adult rats.

Resident microglia are involved in immune responses of the central nervous system and may contribute to neuronal degeneration and death. Here, we tested in adult rats whether injection of bacterial lipopolysaccharide (which causes inflammation and microglial activation) just above the substantia nigra, results in the death of dopaminergic substantia nigra pars compacta neurons. Two weeks after lipopolysaccharide injection, microglial activation was evident throughout the nigra and the number of retrogradely-labeled substantia nigra neurons was reduced to 66% of normal. This suggests that inflammation and/or microglial activation can lead to neuronal cell death in a well-defined adult animal model. The opioid receptor antagonist naloxone reportedly reduces release of cytotoxic substances from microglia and protects cortical neurons in vitro. Here, a continuous two-week infusion of naloxone at a micromolar concentration close to the substantia nigra, prevented most of the neuronal death caused by lipopolysaccharide, i.e. 85% of the neurons survived. In addition, with systemic (subcutaneous) infusion of 0. 1mg/d naloxone, 94% of the neurons survived. Naloxone infusions did not obviously affect the morphological signs of microglial activation, suggesting that naloxone reduces the release of microglial-derived cytotoxic substances. Alternatively, microglia might not cause the neuronal loss, or naloxone might act by blocking opioid receptors on (dopaminergic or GABAergic) neurons.Thus, local inflammation induces and the opioid antagonist naloxone prevents the death of dopaminergic substantia nigra neurons in adult rats. This may be relevant to the understanding of the pathology and treatment of Parkinson's disease, where these neurons degenerate.

BDNF is needed for postnatal maturation of basal forebrain and neostriatum cholinergic neurons in vivo.

Neurotrophins regulate survival, neurite outgrowth, and phenotypic maturation of developing neurons. Brain-derived neurotrophic factor (BDNF) can promote the survival of developing cholinergic forebrain neurons in vitro and reduce their degeneration following injury in adult rats. We investigated the role of endogenous BDNF during postnatal development of these cholinergic neurons by analyzing homozygous BDNF-deficient (-/-) mice and their littermates (+/+, +/-). At P6, the number of choline acetyltransferase- (ChAT) positive neurons in the medial septum was approximately 23% lower in BDNF-/- mice, although their brain and body weight was normal. At P15, control (+/+) littermates had approximately 45% more and approximately 45% larger ChAT-positive neurons and a much denser cholinergic hippocampal innervation than at P6, indicative of maturation of the septohippocampal system. In BDNF-/- mice, the number, size, and ChAT-immunostaining intensity of the cholinergic neurons remained the same between P6 and P15 (few mice survive longer). BDNF-/- mice had about three times more TUNEL-labeled (a marker of apoptosis) cells in the medial septum at P6, consistent with (but not proof of) the possibility that the cholinergic neurons were dying. The cholinergic hippocampal innervation in BDNF-/- mice expanded to a lesser extent than in controls and had reduced levels of acetylcholinesterase staining at P15. The developmental deficits were largely similar in the neostriatum of BDNF-/- mice. These findings suggest that BDNF is critical for postnatal development and maturation of cholinergic forebrain neurons.

p75(NGFR) and cholinergic neurons in the developing forebrain: a re-examination.

The low-affinity nerve growth factor receptor (p75(NGFR)) apparently can mediate apoptosis in a variety of cells in vitro and in vivo. Previously, our laboratory suggested that p75(NGFR) induced apoptosis in a subpopulation of cholinergic forebrain neurons during postnatal development, i.e., the number of choline acetyltransferase (ChAT)-positive neurons in a control strain of mice decreased whereas it remained higher in p75(NGFR)-deficient (-/-) mice. Discrepancies with subsequent data sets in our laboratory caused us to thoroughly re-analyze the fate of these cholinergic medial septum and neostriatal neurons in new sets of p75(NGFR) -/- and two DNA control strains of mice during development. Between postnatal day (P)6 and P15 the number of ChAT-positive neurons detected in the medial septum of 129/Sv mice and Balb/c mice increased by approximately 64% and approximately 62%, respectively. This increase is contrary to previous reports from our laboratory and indicative of normal postnatal development (including an increase in ChAT-enzyme) of the cholinergic forebrain neurons. In p75(NGFR) -/- mice the number of ChAT-positive neurons in the medial septum remained constant between P6 and P15 and was approximately 31% and approximately 56% higher at P6 than 129/Sv and Balb/c mice, respectively. At P15 and adulthood, p75(NGFR) -/- mice had similar numbers of cholinergic neurons as control mice. In the developing neostriatum, the number of ChAT-positive neurons increased by approximately 56% between P6 and P15 and did not differ between p75(NGFR) -/- and control mice at any time. Analyses for apoptotic DNA fragmentation (TUNEL labeling) at P8 revealed no differences between p75(NGFR) -/- and control mice in 12 forebrain regions, including the septum and neostriatum. At all times, all mice had similar levels of acetylcholinesterase-positive cholinergic innervation of the molecular layer in the dorsal dentate gyrus. These findings suggest that the p75(NGFR) does not necessarily mediate apoptosis in medial septum or neostriatal cholinergic neurons during the postnatal time period. The discrepant results of the previous study are most likely due to a less rigorous application of criteria for data acquisition, including anatomical boundaries that define the nucleus.

Neurotrophins promote regeneration of sensory axons in the adult rat spinal cord.

We have investigated the effects of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) on the intraspinal regeneration of anterogradely labeled axotomized ascending primary sensory fibers in the adult rat. These fibers were allowed to grow across a predegenerated peripheral nerve graft and back into the thoracic spinal cord. In control animals that had been infused with vehicle for two weeks into the dorsal column, 3 mm rostral to the nerve graft, essentially no fibers had extended from the nerve graft back into the spinal cord. The number of sensory fibers in the rostral end of the nerve graft was not significantly different between control and neurotrophin-infused animals. With infusion of NGF, 37+/-2% of the fibers at the rostral end of the graft had grown up to 0.5 mm into the dorsal column white matter, 30+/-2% up to 1 mm, 19+/-3% up to 2 mm and 8+/-2% up to 3 mm, i.e., the infusion site. With infusion of NT-3, sensory fiber outgrowth was similar to that seen with NGF, but with BDNF fewer fibers reached farther distances into the cord. Infusion of a mixture of all three neurotrophins did not increase the number of regenerating sensory fibers above that seen after infusion of the individual neurotrophins. These findings suggest that injured ascending sensory axons are responsive to all three neurotrophins and confirm our previous findings that neurotrophic factors can promote regeneration in the adult central nervous system.

P75 nerve growth factor receptor is important for retrograde transport of neurotrophins in adult cholinergic basal forebrain neurons.

The role of the p75 nerve growth factor receptor in the retrograde transport of neurotrophins in the adult CNS was investigated by comparing the transport of 125I-labeled neurotrophins by normal and p75 nerve growth factor receptor-deficient cholinergic septohippocampal neurons. In control mice, nerve growth factor was selectively transported from the hippocampal formation to the cholinergic neurons in the septum. Nerve growth factor labeling was found in three to four times as many septal cholinergic neuronal cell bodies than labeling for neurotrophin-3 or neurotrophin-4/5, and transported brain-derived neurotrophic factor was barely detectable. Cells were considered as labeled when the number of grains per cell exceeded five times background. In p75 nerve growth factor receptor-deficient mice, the number of cholinergic neurons labeled with each of the neurotrophins was reduced by 85-95%. Retrograde labeling of septohippocampal neurons with Fluorogold was not obviously reduced in p75 nerve growth factor receptor-deficient mice, suggesting that general transport mechanisms were not impaired. Despite the reduced neurotrophin transport, cholinergic neurons of p75 nerve growth factor receptor-deficient mice were larger than controls and had an apparently normal density of immunostaining for choline acetyltransferase. Since nerve growth factor is reportedly involved in size regulation and choline acetyltransferase expression, this raises the possibility that the retrograde transport itself is not essential for these events. Thus, p75 nerve growth factor receptor plays an important, although not exclusive, role in the transport of neurotrophins by cholinergic basal forebrain neurons, and retrograde transport of nerve growth factor may not be needed for regulating certain cellular processes.

p75NGFR mediates death of cholinergic neurons during postnatal development of the neostriatum in mice.

We have previously shown that p75 nerve growth factor receptor (p75NGFR) mediates apoptosis of approximately 25% of the cholinergic basal forebrain neurons in normal control mice between postnatal day 6 and 15, but only of cholinergic neurons that lacked the nerve growth factor receptor TrkA. Here, we investigated whether and when the cholinergic neurons of the neostriatum, which express TrkA and p75NGFR during early postnatal times, undergo p75NGFR-mediated death. The cholinergic neurons in the lateral neostriatal regions expressed choline acetyltransferase (ChAT) earlier (postnatal day 3-6) than those of the medial regions and TrkA appeared before ChAT in all regions. Between postnatal day 6 and 10, approximately 40% of the ChAT-positive neurons in the most lateral regions disappeared in control mice but not in p75NGFR-deficient mice. During this time, the neostriatum of control, but not p75NGFR-deficient, mice contained many apoptotic cells. This suggests that, similar to the cholinergic neurons of the basal forebrain, the neostriatal cholinergic neurons of control mice die and that this process is mediated by p75NGFR. However, the roles of p75NGFR and TrkA appear to be more complicated in the neostriatum where relatively few neurons express p75NGFR during the death phase (and predominantly in the lateral neostriatum where the neuronal loss is greatest), and TrkA-positive as well as TrkA-negative neurons may be lost.