Alex Krüttgen

The small GTPase Rab7 controls the endosomal trafficking and neuritogenic signaling of the nerve growth factor receptor TrkA

Nerve growth factor (NGF) and its TrkA receptor exert important bioactivities on neuronal cells such as promoting survival and neurite outgrowth. Activated TrkA receptors are not only localized on the cell surface but also in signaling endosomes, and internalized TrkA receptors are important for the mediation of neurite outgrowth. The regulation of the endosomal trafficking of TrkA is so far unknown. Because the endosome-associated GTPase Rab7 coimmunoprecipitated with TrkA, we examined whether the endosomal trafficking of TrkA might be under the control of Rab7. Inhibiting Rab7 by expression of a green fluorescent protein-tagged, dominant-negative Rab7 variant resulted in endosomal accumulation of TrkA and pronounced enhancement of TrkA signaling in response to limited stimulations with NGF, such as increased activation of Erk1/2 (extracellular signal-regulated kinase 1/2), neurite outgrowth, and expression of GAP-43 (growth-associated protein 43). Our studies show that the endosomal GTPase Rab7 controls the endosomal trafficking and neurite outgrowth signaling of TrkA. Because mutations of Rab7 are found in patients suffering from hereditary polyneuropathies, dysfunction of Rab7 might contribute to neurodegenerative conditions by affecting the trafficking of neurotrophins. Moreover, strategies aimed at controlling Rab7 activity might be useful for the treatment of neurodegenerative diseases.

Structure-function analysis of human interleukin-6 : evidence for the involvement of the carboxy-terminus in function

C‐terminally deleted analogs of human interleukin‐6 (IL‐6) have been constructed at the cDNA level, and after cell‐free transcription and translation their biological activity was analyzed. Removal of only 4 amino acids resulted in complete loss of biological activity as determined by the B9 cell proliferation assay. Secondary structure prediction of human IL‐6 resulted in 58% helix, 14% β‐structure, and 28% turn and coil (average of 3 independent methods). The circular dichroism of recombinant human IL‐6 was measured in the near and far UV. Evaluation of the latter in terms of secondary structures gave 67% helix, 15% β‐structure, and 18% turn and coil.

The Dark Side of the NGF Family: Neurotrophins in Neoplasias

Although neurotrophins of the nerve growth factor (NGF) family are best known for their neurite outgrowth‐inducing and survival‐promoting effects on neuronal cells, these are actually pleiotropic growth factors acting physiologically on many different cell types of our body. As for many other growth factors, dysregulation of neurotrophin signal transduction is found in a number of tumors where they can accompany or contribute to malignant transformation. Interestingly, tropomyosin‐related kinase (Trk) receptor activation can either support or suppress tumor growth, depending on the tumor type. These same divergent responses have been observed with neurotrophins binding to the p75NTR neurotrophin receptor on tumor cells. This article summarizes the current knowledge on the role of neurotrophins and their receptors in malignancies, with special focus on tumors of neuropathological interest.

Signalling pathways leading to neuroblastoma differentiation after serum withdrawal: HDL blocks neuroblastoma differentiation by inhibition of EGFR.

Neuroblastoma is the second most common pediatric malignancy, characterized by a high rate of unexplained spontaneous remissions. Much progress has been made in understanding neuroblastoma differentiation triggered by certain agents such as retinoic acid. However, little is known about the signalling pathways that lead to differentiation of neuroblastoma cells due to serum withdrawal. We found that in Neuro2a neuroblastoma cells, EGFR, ERK1/2 and Akt showed increased phosphorylation after serum withdrawal, and that the activation of EGFR was necessary for the activation of Akt and ERK1/2. Inhibition of EGFR, ERK1/2 and PI3K blocked neuroblastoma differentiation after serum withdrawal. Interestingly, addition of high-density lipoprotein (HDL) abrogated serum-withdrawal induced neuroblastoma differentiation, as well as the activation of EGFR. Our results demonstrate a novel role for serum-derived lipoproteins in the control of receptor tyrosine kinase activity.

Differential endocytic sorting of p75NTR and TrkA in response to NGF: a role for late endosomes in TrkA trafficking

NGF binds to two receptors, p75NTR and TrkA. The endosomal trafficking of receptors is of emerging importance for the understanding of their signaling. We compared the endocytic trafficking of the two NGF receptors in PC12 cells. Both p75NTR and TrkA were internalized in response to NGF and colocalized with early endosomes. However, surprisingly, the subsequent endosomal trafficking paths of both NGF receptors diverged: whereas p75NTR recycled back to the surface, TrkA moved to late endosomes and underwent lysosomal degradation. By performing subcellular fractionations of NGF stimulated PC12 cells, tyrosine-phosphorylated TrkA was recovered in fractions corresponding to late endosomes. This implicates these organelles as novel endosomal NGF signaling platforms. Furthermore, the trafficking of NGF receptors could be manipulated by pharmacological means. Disrupting p75NTR recycling diminished TrkA activation in response to low concentrations of NGF, demonstrating a functional role for the recycling of p75NTR.

The three carboxy-terminal amino acids of human interleukin-6 are essential for its biological activity

We have constructed on the cDNA level deletion mutants of human interleukin‐6 lacking one, two, three or four amino acids from the carboxyterminus of the molecule. After in vitro transcription and translation the biological activity of these deletion mutants was determined by two independent bioassays. Both, the mouse B9 cell proliferation assay and the fibrinogen induction assay with the human hepatoma cell line HepG2 led to the following result: already the removal of the last amino acid resulted in a five‐fold loss of biological activity. An additional slight reduction was seen when two amino acids were removed from the carboxy‐terminus. Interleukin‐6 lacking three or four C‐terminal amino acids were completely inactive. The presented results emphasize the extreme importance of the carboxy‐terminus of interleukin‐6 for its biological function.

The secreted brain-derived neurotrophic factor precursor pro-BDNF binds to TrkB and p75NTR but not to TrkA or TrkC.

The neurotrophin brain‐derived neurotrophic factor (BDNF) binds to two cell surface receptors: TrkB receptors that promote neuronal survival and differentiation and p75NTR that induces apoptosis or survival. BDNF, as well as the other members of the neurotrophin family, is synthesized as a larger precursor, pro‐BDNF, which undergoes posttranslational modifications and proteolytic processing by furin or related proteases. Both mature neurotrophins and uncleaved proneurotrophins are secreted from cells. The bioactivities of proneurotrophins could differ from those of mature, cleaved neurotrophins; therefore, we wanted to test whether pro‐BDNF would differ from mature BDNF in its neurotrophin receptor binding and activation. A furin‐resistant pro‐BDNF, secreted from COS‐7 cells, bound to TrkB‐Fc and p75NTR‐Fc, but not to TrkA‐Fc or TrkC‐Fc. Likewise, pro‐BDNF elicited prototypical TrkB responses in biological assays, such as TrkB tyrosine phosphorylation, activation of ERK1/2, and neurite outgrowth. Moreover, mutation of the R103 residue of pro‐BDNF abrogated its binding to TrkB‐Fc but not to p75NTR‐Fc. Taken together, these data indicate that pro‐BDNF binds to and activates TrkB and could be involved in TrkB‐mediated neurotrophic activity in vivo.

Nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 are sorted to dense-core vesicles and released via the regulated pathway in primary rat cortical neurons

Neurotrophins (NTs) play an important role in the modulation of synaptic transmission and in morphological changes in synaptic structures. Although there is agreement that brain‐derived neurotrophic factor (BDNF) is sorted to large dense‐core vesicles (LDCVs) and released via the regulated secretory pathway, there has been some dispute regarding the mode of secretion of nerve growth factor (NGF) and neurotrophin‐3 (NT‐3), two structurally related members of the NT family. In this study, we examined the subcellular localization and release characteristics of NGF, BDNF, and NT‐3 in adenovirus‐infected primary cortical neurons. We found that all members of the NT family colocalized with markers for the endoplasmic reticulum and Golgi within cell bodies and in a punctate manner with a marker for LDCVs within processes. Moreover, their release was triggered by depolarization, indicating that NGF, BDNF, and NT‐3 are released via the regulated secretory pathway. When neurons were coinfected with two separate adenoviruses coding for NGF or BDNF, both NTs showed almost complete vesicular colocalization within single cells, suggesting that different NTs might be packaged into shared vesicles. We also examined whether the two splice variants of NGF, the short and long precursors, differ in their release characteristics. We found that neurons infected with viruses coding for either splice variant released NGF in a regulated way. Overall, our study supports the notion that all members of the NT family undergo activity‐dependent regulated release from neurons, enabling them to act as “synaptotrophins” on electrically active neurons.

CMT2B-associated Rab7 mutants inhibit neurite outgrowth

Charco–Marie–Tooth type 2B (CMT2B) neuropathy is a rare autosomal-dominant axonal disorder characterized by distal weakness, muscle atrophy, and prominent sensory loss often complicated by foot ulcerations. CMT2B is associated with mutations of the Rab7 protein, a small GTPase controlling late endocytic traffic. Currently, it is still unknown how these mutations cause the neuropathy. Indeed, CMT2B selectively affects neuronal processes, despite the ubiquitous expression of Rab7. Therefore, this study focused on whether these disorder-associated mutations exert an effect on neurite outgrowth. We observed a marked inhibition of neurite outgrowth upon expression of all the CMT2B-associated mutants in the PC12 and Neuro2A cell lines. Thus, our data strongly support previous genetic data which proposed that these Rab7 mutations are indeed causally related to CMT2B. Inhibition of neurite outgrowth by these CMT2B-associated Rab7 mutants was confirmed biochemically by impaired up-regulation of growth-associated protein 43 (GAP43) in PC12 cells and of the nuclear neuronal differentiation marker NeuN in Neuro2A cells. Expression of a constitutively active Rab7 mutant had a similar effect to the expression of the CMT2B-associated Rab7 mutants. The active behavior of these CMT2B-associated mutants is in line with their previously demonstrated increased GTP loading, thus confirming that active Rab7 mutants are responsible for CMT2B. Our findings provide an explanation for the ability of CMT2B-associated Rab7 mutants to override the activity of wild-type Rab7 in heterozygous patients. Thus, our data suggest that lowering the activity of Rab7 in neurons could be a targeted therapy for CMT2B.

Evidence for the importance of a positive charge and an α-helical structure of the C-terminus for biological activity of human IL-6

Thirteen point mutations of human interleukin‐6 at the C‐terminus were constructed at the cDNA‐level. Two degenerate oligonucleotide primers were used for PCR synthesis of two groups of point mutations at positions 182 and 184 The mutated cDNAs were in vitro transcribed and translated and subsequently assayed for biological activity in the B9 cell proliferation test. Our results confirm our former findings obtained with deletion mutants on the importance of the C‐terminus of IL‐6 for biological activity. In addition, we now present evidence for the importance of an α‐helix at the C‐terminus of IL‐6 and the presence of the positive charge at position 182 for biological activity.