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.

Survival of cholinergic forebrain neurons in developing p75NGFR-deficient mice.

The functions of the low-affinity p75 nerve growth factor receptor (p75(NGFR)) in the central nervous system were explored in vivo. In normal mice, approximately 25 percent of the cholinergic basal forebrain neurons did not express TrkA and died between postnatal day 6 and 15. This loss did not occur in p75(NGFR)-deficient mice or in normal mice systemically injected with a p75(NGFR)-inhibiting peptide. Control, but not p75(NGFR)-deficient, mice also had fewer cholinergic striatal interneurons. Apparently, p75(NGFR) mediates apoptosis of these developing neurons in the absence of TrkA, and modulation of p75(NGFR) can promote neuronal survival. Cholinergic basal forebrain neurons are involved in learning and memory.

Laminin-like antigen in rat CNS neurons: distribution and changes upon brain injury and nerve growth factor treatment.

Using several antibodies against rat or human laminin and an avidin-biotin immunocytochemical protocol, laminin-like immunoreactivity was detectable in the rat nervous system in expected locations, i.e., associated with blood vessels and reactive astrocytes. However, laminin staining was also abundantly present within neuronal cell bodies in most parts of the developing and adult rat CNS. Medial septum neuronal immunoreactivity was lost after septo-hippocampal disconnection, but could be preserved or even restored by intraventricular administration of nerve growth factor. Thus, at least for medial septum neurons, this laminin-like molecule can be accumulated or produced independent of direct hippocampal (target) contact. It remains to be determined whether CNS neuronal "laminin" processes activities similar to those found for laminin in vitro.

Ciliary neurotrophic factor prevents neuronal degeneration and promotes low affinity NGF receptor expression in the adult rat CNS.

Recombinant human ciliary neurotrophic factor (CNTF) was infused for 2 weeks into the lateral ventricle of fimbria-fornix transected adult rats, and its effects were compared with those of purified mouse nerve growth factor (NGF). We provide evidence that CNTF can prevent degeneration and atrophy of almost all injured medial septum neurons (whereas NGF protects only the cholinergic ones). CNTF is also involved in up-regulation of immunostainable low affinity NGF receptor (LNGFR) in cholinergic medial septum and neostriatal neurons and in a population of lateral septum neurons. In contrast to NGF, CNTF did not stimulate choline acetyltransferase in the lesioned septum and normal neostriatum (pointing to different mechanisms for the regulation of choline acetyltransferase and LNGFR), cause hypertrophy of septal or neostriatal cholinergic neurons, or cause sprouting of LNGFR-positive (cholinergic) septal fibers.

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.

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).

Glial cell line-derived neurotrophic factor prevents death, but not reductions in tyrosine hydroxylase, of injured nigrostriatal neurons in adult rats.

Glial cell line-derived neurotrophic factor (GDNF) promotes survival of mesencephalic dopaminergic neurons in vitro and when injected locally into the brains of lesioned adult animals. Here, we show that GDNF (3 micrograms per day and higher) can promote the survival of all (retrogradely labeled) axotomized nigrostriatal dopaminergic neurons of adult rats when continuously infused for 2 weeks close to the substantia nigra, compared to only approximately 30% survival with control infusions. Based on our previous observations, GDNF was as potent as ciliary neurotrophic factor and neurotrophin-4 and approximately five to ten times more potent than brain-derived neurotrophic factor and was most effective in promoting survival. GDNF prevented neuronal death induced by 6-hydroxydopamine to a lesser extent than after axotomy. GDNF treatments begun 1 week after axotomy could maintain those neurons that had not yet died. When a 2 week GDNF treatment was interrupted, most of the GDNF-rescued neurons died over the following 2 weeks. This suggests that longer trophic factor treatments or nigrostriatal connections are needed to achieve permanent survival. Measurements of tyrosine hydroxylase (TH) immunoreactivity of the rescued neuronal cell bodies suggest that GDNF cannot prevent the lesion-induced loss of this rate-limiting enzyme for dopamine synthesis. In fact, GDNF induced a decrease in TH in normal animals, suggesting an active down-regulation of TH synthesis. Levels of TH immunoreactivity were recovered between 7 and 14 days after withdrawal of a 2 week GDNF infusion, in the neurons that survived axotomy. These results may have implications for developing new treatment strategies for Parkinson's disease.

Distribution of corticospinal motor neurons in the postnatal rat: quantitative evidence for massive collateral elimination and modest cell death.

The postnatal development of rat corticospinal motor neurons (CSMN) was studied by retrograde tracing with cholera toxin B subunit (CTB) injected into the upper cervical dorsal spinal cord on the first postnatal day (P0), P3, P10, P20, and at adulthood. CTB-labeled neurons were visualized by immunocytochemistry and extensively quantified throughout the cortex. At P0, CSMN were found to an extent similar to that reported in P3 animals with other neuronal tracers, now permitting in vitro studies of neonatal CSMN. Between P0 and P3, the number of labeled neurons increased by 30% to a total maximum of approximately 185,000 in both cortices. The increase occurred throughout the cortex. At P10, the number of labeled CSMN had decreased to 60% of the number at P3. Fewer CSMN were evident particularly in the perirhinal cortex. Between P10 and P20, the number of CSMN decreased further to 52% of the maximal number at P3. This decrease occurred predominantly in the cingulate and parietal cortex. The number of labeled CSMN in rats injected at P0 and analyzed at P20 was 10% lower than the number in P0-injected littermates that were analyzed at P3, which suggests that only a small portion of the "disappearing" CSMN undergoes developmental neuronal death. Thus, the spinal projection of the remaining 38% is apparently eliminated between P3 and P20. Detailed quantitative analysis of the CSMN distribution demonstrated that neuronal death occurs predominantly in the perirhinal cortex. In contrast, axonal elimination of corticospinal projections occurred throughout the CSMN field, i.e., primarily in the frontal, occipital, and perirhinal cortex between P3-P10 and in the cingulate and parietal cortex between P10-P20.

Nerve growth factor receptor immunoreactivity in neurons of the normal adult rat spinal cord and its modulation after peripheral nerve lesions.

Motoneurons of the rat spinal cord express low-affinity nerve growth factor receptor (LNGFR) and corresponding mRNA during development, and re-express it after their axotomy by peripheral nerve injury. The present study establishes the anatomical and quantitative baseline of LNGFR immunoreactive (LNGFR-IR) neurons of the entire normal adult female rat and then investigates the temporal course for the re-expression of LNGFR-IR in lumbar motoneurons after either a crush lesion (which is followed by regeneration and reconnection to the muscle) or a cut lesion with removal of the distal stump (where a neuroma but no reconnection is formed). In the normal adult spinal cord, two types of LNGFR-IR neurons were recognized: (1) small populations of large motoneurons located in the ventral horn mainly in correspondence to the regions of the phrenic, cremasteric and dorsolateral nuclei, and (2) a more numerous and more dorsally located population of small neurons. With a sciatic cut lesion, the number of LNGFR-IR motoneurons at spinal levels L4-L6 rapidly and dramatically increased to a maximum between post-lesion days 1 and 7, apparently involving most axotomized motoneurons of the region, and returned to the baseline level by day 30. With a crush lesion, similar numbers and virtually the same time-course of LNGFR-IR appearance were seen, but the onset of progressive disappearance of LNGFR-IR neurons was delayed by one week, so that at 30 days, the most caudal motoneurons (which are last to reach their target) were still LNGFR-IR. Comparison of these two time courses gives clues to the kind of signals that may be involved in initiating and/or maintaining the LNGFR response.

The localization of nerve growth factor-like immunoreactivity in the adult rat basal forebrain and hippocampal formation.

The role of nerve growth factor (NGF) as a target derived neurotrophic agent for specific cell populations in the peripheral nervous system has been well documented and much evidence suggests that NGF may serve a similar neurotrophic role in the CNS supporting the cholinergic neurons of the basal forebrain. Previous attempts to localize NGF by immunocytochemical methods, however, have not yielded evidence confirming the regional distribution expected based upon reported levels of extractable NGF. In the present study, affinity purified polyclonal antibodies to beta-NGF and a modified immunohistochemical protocol were used to demonstrate specific NGF-like immunoreactivity in the adult rat hippocampal formation and basal forebrain. In the hippocampal formation, NGF-like immunoreactivity was localized primarily within the hilus of the dentate gyrus and within stratum lucidum of the CA3 and CA2 hippocampal subfields. Staining appeared to be associated with cell processes and was similar to the reported distribution of mossy fibers suggesting that granule cells may either serve as a primary source of hippocampal NGF or that mossy fibers selectively accumulate NGF produced by other cell populations. In the basal forebrain, NGF-like immunoreactivity was localized within neuronal cell bodies of the medial septum, diagonal band, and nucleus basalis of Meynert and was further demonstrated to colocalize exclusively with LNGF-R positive neurons. These findings demonstrate the presence of an NGF-like antigen in association with cholinergic neurons of the basal forebrain and strongly support the hypothesis that NGF may serve as an endogenous trophic factor for this adult neuronal population.

Ciliary neurotrophic factor (CNTF) promotes low-affinity nerve growth factor receptor and CD4 expression by rat CNS microglia.

Ramified parenchymal microglia may provide immune surveillance in the nervous system and become activated in response to injury, showing increases in antigens found on macrophages, e.g. CD4 and MHCs. We investigated in adult rats the effects of a 2-week intraventricular infusion with ciliary neurotrophic factor (CNTF), a nervous system-associated cytokine, on microglia of the normal and injured corpus callosum. CNTF caused morphological changes, induced the expression of low-affinity nerve growth factor receptor and CD4 and increased the expression of complement receptor 3. Such changes were also observed after treatment of pure cultures of neonatal rat microglial cells with highly purified CNTF, suggesting a direct responsiveness to CNTF. Thus, endogenous astroglial and Schwann cell-derived CNTF may be an important component of the immune responses of the nervous system.

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.

Nerve growth factor effects on cholinergic neurons of neostriatum and nucleus accumbens in the adult rat.

Following intraventricular nerve growth factor infusion in adult rats, the choline acetyltransferase immunostaining of the neuropil and neuronal cell bodies of the neostriatum (caudate-putamen) and nucleus accumbens was more intense on the side of the infusion. Furthermore, the average cross-sectional size (micron2) of the cholinergic somata was increased by about 40 and 20% in the striatum and accumbens, respectively. This unilateral response could be elicited in intact rats as well as in rats receiving a prior aspirative transection of the fimbria-fornix. The reported lack of (low-affinity) nerve growth factor receptor immunostaining in these neurons suggests that the nerve growth factor effects are most likely transduced by high-affinity receptors. The ability of these apparently undamaged cholinergic interneurons to respond to exogenous nerve growth factor with an increase in choline acetyltransferase content and cell body size suggests that they are benefiting from a less-than-maximal support by endogenous nerve growth factor in the normal young adult rat.

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.

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.

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.

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.

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.