Alzheimer's disease and NGF signaling.

Age-related degeneration of basal forebrain cholinergic neurons (BFCNs) occurs early and contributes significantly to cognitive decline in Alzheimer's disease (AD). Proper function and morphology of BFCNs depends on the supply of nerve growth factor (NGF) from the cortex and the hippocampus. A large number of experiments have shown that decreased supply of NGF at the level of BFCN cell bodies leads to loss of neuronal markers and shrinkage, mimicking what is observed in AD. The delivery of sufficient amounts of NGF signal to BFCN cell bodies depends on the effective participation of several factors including sufficient synthesis and release of NGF, adequate synthesis and expression of NGF receptors by BFCNs, normal signaling and retrograde transport of NGF-receptor complex, and finally effective induction of gene expression by NGF. In the past few years it has become clear that decreased amounts of NGF at the level of BFCN cell bodies is largely due to failed retrograde transport rather than decreased synthesis, binding or expression of NGF receptors in the BFCN terminals. We will discuss in vivo evidence supporting decreased retrograde transport of NGF in a mouse model with BFCN degeneration, and will attempt to match these findings with our studies in postmortem human AD brain. We will speculate about the possible mechanisms of failed NGF retrograde transport and its relationship to AD pathology.

Enhanced activation of axonally transported stress-activated protein kinases in peripheral nerve in diabetic neuropathy is prevented by neurotrophin-3.

The objective was to determine whether stress-activated protein kinases (SAPKs) mediated the transfer of diabetes-induced stress signals from the periphery to somata of sensory neurons. Thus, we characterized axonal transport of SAPKs in peripheral nerve, studied any alteration in streptozotocin (STZ)-diabetic rats and examined effects of neurotrophin-3 (NT-3) on diabetes-induced events. We demonstrate that c-jun N-terminal kinase (JNK) and p38 are bidirectionally axonally transported at fast rates in sciatic nerve. In STZ-diabetic rats the relative levels of retrograde axonal transport of phosphorylated (activated) JNK and p38 were raised compared with age-matched controls (all data are in arbitrary units and expressed as fold increase over control: JNK 54-56 kDa isoforms, control 1.0 +/- 0.19, diabetic 2.5 +/- 0.26; p38, control 1.0 +/- 0.09, diabetic 2.9 +/- 0.52; both P < 0.05). Transport of total enzyme levels of JNK and p38 and phosphorylated extracellular signal-regulated kinase (ERK) was not significantly altered and anterograde axonal transport of phosphorylated JNK and p38 was unaffected by diabetes. The transcription factor ATF-2, which is phosphorylated and activated by JNK and p38, also exhibited elevated retrograde axonal transport in STZ-diabetic animals (control 1.0 +/- 0.07, diabetic 3.0 +/- 0.41; P < 0.05). Treatment of STZ-diabetic animals with 5 mg/kg human recombinant NT-3 prevented activation of JNK and p38 in sciatic nerve (phosphorylated JNK, control 1.0 +/- 0.09, diabetic 1.95 +/- 0.35, diabetic + NT-3 1.09 +/- 0.12; P < 0.05 diabetic versus others; phosphorylated p38, control 1.0 +/- 0.16, diabetic 4.7 +/- 0.9, diabetic + NT-3 1.19 +/- 0.18; P < 0.05 diabetic versus others). The results show that JNK and p38 are transported axonally and may mediate the transfer of diabetes-related stress signals, possibly triggered by loss of neurotrophic support, from the periphery to the neuronal soma.

Nerve growth factor modulates the activation status and fast axonal transport of ERK 1/2 in adult nociceptive neurones.

Mature dorsal root ganglion cells respond to neurotrophins, and the intracellular signalling pathways activated by neurotrophins have been characterized in vitro. We have now used immunocytochemistry and Western blots to examine the expression and activation of extracellular signal-regulated protein kinase-1/2 (ERK) in rat dorsal root ganglion cells in vivo, using antisera to total (tERK) and phosphorylated (pERK) forms. This has revealed a number of novel findings. tERK immunoreactivity is present in most dorsal root ganglion cells but is expressed most strongly in small (nociceptive) cells and, surprisingly, is absent in a population of large cells that expressed trkB or trkC but mainly lack p75(NTR) immunoreactivity. In contrast pERK is prominent in a few trkA cells and in satellite glial cells, and is further increased by NGF treatment. tERK and pERK both undergo fast anterograde and retrograde axonal transport, indicated by accumulation at a sciatic nerve ligature, and NGF reduces the level of retrograde pERK transport.

Failed retrograde transport of NGF in a mouse model of Down's syndrome: reversal of cholinergic neurodegenerative phenotypes following NGF infusion.

Age-related degeneration of basal forebrain cholinergic neurons (BFCNs) contributes to cognitive decline in Alzheimer's disease and Down's syndrome. With aging, the partial trisomy 16 (Ts65Dn) mouse model of Down's syndrome exhibited reductions in BFCN size and number and regressive changes in the hippocampal terminal fields of these neurons with respect to diploid controls. The changes were associated with significantly impaired retrograde transport of nerve growth factor (NGF) from the hippocampus to the basal forebrain. Intracerebroventricular NGF infusion reversed well established abnormalities in BFCN size and number and restored the deficit in cholinergic innervation. The findings are evidence that even BFCNs chronically deprived of endogenous NGF respond to an intervention that compensates for defective retrograde transport. We suggest that age-related cholinergic neurodegeneration may be a treatable disorder of failed retrograde NGF signaling.

Axonal transport of activating transcription factor-2 is modulated by nerve growth factor in nociceptive neurons.

The aim of this study was to determine whether axonal transport of activating transcription factor-2 (ATF2) occurs in adult sensory neurons, and whether this process is under neurotrophin control. Antisera to both total ATF2 and to the activated (i.e., phosphorylated) form were used for immunocytochemistry and Western blotting. ATF2 was localized to predominantly nociceptive dorsal root ganglion cells in adult rats and shown to accumulate proximal and distal to a sciatic nerve ligature as a result of axonal transport. Subcutaneous injection of nerve growth factor (NGF) decreased the levels of fast retrograde axonal transport of activated ATF2 by 97% (p < 0.05) and elevated levels of retrograde axonal transport of total ATF2 by twofold (p < 0.02). In contrast, blocking endogenous NGF using an anti-NGF antibody induced an elevation in retrograde axonal transport of activated ATF2 of 4. 5-fold (p < 0.05) and decreased retrograde axonal transport of total ATF2 by 72% (p < 0.05). NGF or anti-NGF treatment had no effect on the anterograde transport levels of total or activated ATF2. This study shows that signaling by target-derived NGF to the cell bodies of sensory neurons consists, in part, of the modulation of levels and activation status of a retrogradely transported transcription factor, ATF2.

Focally administered nerve growth factor suppresses molecular regenerative responses of axotomized peripheral afferents in rats.

Effects of delivery of nerve growth factor, from a catheterized osmotic mini-pump to the proximal stump of a transected sciatic nerve, were compared with the effects of normal saline. A pilot measured retrograde axonal transport of nerve growth factor to determine a pump concentration which raised axonal transport ipsilaterally, but not contralaterally. The effects of this delivery over 12 days were then determined on expression of growth-associated protein-43, trkA, p75NTR and preprotachykinin A ipsilateral and contralateral to the pump in dorsal root ganglia at L4 and L5 (pooled). Ganglionic expression was measured both as messenger RNA and protein. Axotomy (saline pumps) increased growth-associated protein-43 messenger RNA (318 +/- 14%: all changes are percent of contralateral, non-axotomized ganglia with saline pumps) and immunoreactivity (431 +/- 43%). The increase was significantly less (P < 0.001) ipsilateral to nerve growth factor pumps (191 +/- 45%). Axotomy reduced expression of p75NTR (messenger RNA: 52 +/- 17%, P < 0.01; immunoreactivity: 74 +/- 3%, P < 0.05). These decreases were converted to increases by nerve growth factor delivery (respectively 143 +/- 40% and 281 +/- 67%; both P < 0.01). With trkA, axotomy decreased the expression of the messenger RNA (68 +/- 40%, P < 0.01) and of the primary translation product--110,000 mol. wt protein (55 +/- 12%, P < 0.01)--but not the fully glycosylated trkA protein (mol. wt 145,000). Nerve growth factor delivery did not affect trkA expression. Axotomy reduced messenger RNA for the substance P precursor, preprotachykinin A, to 42 +/- 17% (P < 0.01) and this reduction was prevented by nerve growth factor treatment. We suggest that the primary effect of nerve growth factor on axotomized C-fibres is not to promote regeneration, although that may be its secondary effect via an action on Schwann cells. It is possible that reduced neuronal sensitivity to nerve growth factor during regeneration is advantageous in suppressing nociception.

Effect of nerve growth factor treatment on p75NTR gene expression in lumbar dorsal root ganglia of streptozocin-induced diabetic rats.

Previous work shows that gene expression for p75NTR in lumbar dorsal root ganglia (DRG) is deficient in streptozocin-induced diabetic rats, while expression of trkA protein is unaffected. This is the first study on the effect of diabetes on immunohistochemical staining and axonal transport of p75NTR in sensory neurons. We also investigated the novel effect of nerve growth factor (NGF) treatment on the levels of mRNA and protein of the NGF receptors, trkA and p75NTR, in normal and diabetic rats. Immunohistochemical staining for p75NTR was significantly reduced in DRG of 12-week streptozocin-induced diabetic rats, confirming the previous work. Anterograde and retrograde axonal transport of p75NTR within the sciatic nerve was similarly decreased in diabetic rats, while levels of transport of trkA were unaffected. Treatment with systemic NGF reversed the diabetes-induced deficit in p75NTR transcripts and protein within the DRG and left expression levels of trkA unchanged. We propose that in sensory neurons of diabetic rats, the ability to capture and retrogradely transport NGF may be impaired because of suboptimal production of p75NTR receptors and that NGF therapy may overcome this deficiency.

A native electrostatic environment near Q(B) is not sufficient to ensure rapid proton delivery in photosynthetic reaction centers.

Flash-induced absorption spectroscopy has been used to characterize Rhodobacter capsulatus reaction centers mutated in the secondary quinone acceptor site (Q(B). We compared the wild-type, the L212Glu-L213Asp --> Ala-Ala photosynthetically incompetent double mutant (DM), and two photocompetent revertants, the DM+L217Arg --> Cys and the DM+M5Asn- --> Asp strains. The electrostatic environment for Q(B)- is different in the two revertant strains. Only the L217Arg --> Cys mutation nearly restores the native electrostatic environment of Q(B)-. However, the level of recovery of the reaction center function, measured by the rates of second electron transfer and cytochrome c turnover, is quite incomplete in both strains. This shows that a wild-type-like electrostatic environment of Q(B)- cannot ensure on its own, rapid and efficient proton transfer to Q(B)-.

Diabetes and axotomy-induced deficits in retrograde axonal transport of nerve growth factor correlate with decreased levels of p75LNTR protein in lumbar dorsal root ganglia.

The effect of sensory neurone axotomy on the level of retrograde axonal transport of nerve growth factor (NGF) was studied in the sciatic nerve of age-matched normal and 8-week streptozocin-diabetic rats. In normal rats a 10-day sciatic nerve crush induced a 41% decrease in transported NGF, however, axotomy of sensory neurones of diabetic rats did not significantly effect the already deficient levels of NGF undergoing retrograde transport. At first sight, this result indicated that transported NGF levels in the sciatic nerve of diabetic rats are at a residual level due to deficient availability of target-derived NGF. To confirm this, the relationship of the transported NGF to the level of sensory neurone expression of the NGF receptor proteins was analysed. Western blots of L4 and L5 dorsal root ganglia (DRG) homogenates revealed no effect of axotomy and/or diabetes on the levels of the 145-kDa tyrosine kinase form of trkA. However, the expression of p75LNTR protein in the intact DRG was reduced in diabetic compared with normal rats (56%; P < 0.01), and axotomy reduced the levels in the ipsilateral ganglia of normal but not diabetic rats - as seen for NGF axonal transport. Reductions in retrograde axonal transport of NGF in both diabetes and/or axotomy were associated with the levels of p75LNTR within the lumbar DRG.