Apoptosis In Sensory Neurons Research Articles

Persistent SARS‐CoV‐2 Effects Induce Neuropathy Signature in Dorsal Root Ganglia Underlying Hypersensitivity in a Hamster Model

Post‐acute sequelae of COVID‐19, commonly known as long‐COVID, is defined as a persistent symptom(s) that is unexplainable by alternative diagnosis and lasts beyond three months after the onset of COVID‐19. Several studies have now identified long‐COVID in >50% of COVID‐19 patients, emphasizing the need for elucidating mechanisms underlying these symptoms. Pain is a prevalent symptom in long‐COVID patients and presents as headache, persistent muscle pain, joint pain, stomach pain, chest pain, respiratory discomfort, and dysesthesia or paresthesia. Understanding the molecular underpinnings of pain maintenance in these patients could provide information on novel therapeutic strategies. Our lab has previously identified novel treatments for pain due to peripheral inflammation from dysesthesia‐inducing mechanisms associated with acute SARS‐CoV‐2 (SCV2) respiratory infection in hamsters (1‐4 days post‐infection, dpi). We have also previously demonstrated the validity of long‐term infection of hamsters (30‐60 dpi) as a pre‐clinical model of long‐COVID. By utilizing the Von Frey assay, we found that SCV2, but not Influenza A, causes mechanical hypersensitivity at 28 dpi in both male and female hamsters. Bulk RNA sequencing of 31 dpi thoracic dorsal root ganglia (DRGs) revealed a unique transcriptional perturbation signature (168 DEGs, 47 up & 121 down, p‐adj.<0.1), despite viral clearance at approximately 7 dpi. Ingenuity Pathway Analysis of this sequencing (853 DEGs, p‐nom.<0.05) identified several injury‐related canonical pathways, including Production of NO & ROS in Macrophages, Signaling by Rho Family GTPases, mTOR Signaling, Estrogen Receptor Signaling, and Ephrin Receptor Signaling. The Enrichr DisGeNET browser associated these transcriptional changes with clinical neurodegeneration phenotypes, including Amyotrophic Lateral Sclerosis, Alzheimer’s Disease, Neurodegenerative Disorders, and Parkinson Disease. Of note, TUNEL staining did not demonstrate any sign of SCV2‐induced sensory neuron apoptosis at 31 dpi. Further investigation of the sequencing dataset revealed a broad downregulation of TubbmRNA isoforms and Mbp, suggesting microtubule and myelin dysregulation. We also observed a drastic increase in Scn8areads, which could point to Nav1.6‐induced DRG neuron hyperexcitability. In conclusion, our findings suggest that SCV2 leaves a lasting hypersensitivity‐associated transcriptomic signature in DRGs despite early viral clearance that appears to be associated with neurodegeneration mechanisms. As we continue investigating the mechanisms underlying SCV2‐induced actions in DRGs and peripheral nerves, we will also use an upstream regulator analysis of our RNA sequencing data to identify promising therapeutic targets.

Human APOE ɛ3 and APOE ɛ4 Alleles Have Differential Effects on Mouse Olfactory Epithelium.

Alzheimer's disease (AD) is a progressive age-dependent disorder whose risk is affected by genetic factors. Better models for investigating early effects of risk factors such as apolipoprotein E (APOE) genotype are needed. To determine whether APOE genotype produces neuropathologies in an AD-susceptible neural system, we compared effects of human APOE ɛ3 (E3) and APOE ɛ4 (E4) alleles on the mouse olfactory epithelium. RNA-Seq using the STAR aligner and DESeq2, immunohistochemistry for activated caspase-3 and phosphorylated histone H3, glucose uptake after oral gavage of 2-[1,2-3H (N)]-deoxy-D-glucose, and Seahorse Mito Stress tests on dissociated olfactory mucosal cells. E3 and E4 olfactory mucosae show 121 differentially abundant mRNAs at age 6 months. These do not indicate differences in cell type proportions, but effects on 17 odorant receptor mRNAs suggest small differences in tissue development. Ten oxidoreductases mRNAs important for cellular metabolism and mitochondria are less abundant in E4 olfactory mucosae but this does not translate into differences in cellular respiration. E4 olfactory mucosae show lower glucose uptake, characteristic of AD susceptibility and consistent with greater expression of the glucose-sensitive gene, Asns. Olfactory sensory neuron apoptosis is unaffected at age 6 months but is greater in E4 mice at 10 months. Effects of human APOE alleles on mouse olfactory epithelium phenotype are apparent in early adulthood, and neuronal loss begins to increase by middle age (10 months). The olfactory epithelium is an appropriate model for the ability of human APOE alleles to modulate age-dependent effects associated with the progression of AD.

BDNF-TrkB and proBDNF-p75NTR/Sortilin Signaling Pathways are Involved in Mitochondria-Mediated Neuronal Apoptosis in Dorsal Root Ganglia after Sciatic Nerve Transection.

Brain-Derived Neurotrophic Factor (BDNF) plays critical roles during development of the central and peripheral nervous systems, as well as in neuronal survival after injury. Although proBDNF induces neuronal apoptosis after injury in vivo, whether it can also act as a death factor in vitro and in vivo under physiological conditions and after nerve injury, as well as its mechanism of inducing apoptosis, is still unclear. In this study, we investigated the mechanisms by which proBDNF causes apoptosis in sensory neurons and Satellite Glial Cells (SGCs) in Dorsal Root Ganglia (DRG) After Sciatic Nerve Transection (SNT). SGCs cultures were prepared and a scratch model was established to analyze the role of proBDNF in sensory neurons and SGCs in DRG following SNT. Following treatment with proBDNF antiserum, TUNEL and immunohistochemistry staining were used to detect the expression of Glial Fibrillary Acidic Protein (GFAP) and Calcitonin Gene-Related Peptide (CGRP) in DRG tissue; immunocytochemistry and Cell Counting Kit-8 (CCK8) assay were used to detect GFAP expression and cell viability of SGCs, respectively. RT-qPCR, western blot, and ELISA were used to measure mRNA and protein levels, respectively, of key factors in BDNF-TrkB, proBDNF-p75NTR/sortilin, and apoptosis signaling pathways. proBDNF induced mitochondrial apoptosis of SGCs and neurons by modulating BDNF-TrkB and proBDNF-p75NTR/sortilin signaling pathways. In addition, neuroprotection was achieved by inhibiting the biological activity of endogenous proBDNF protein by injection of anti-proBDNF serum. Furthermore, the anti-proBDNF serum inhibited the activation of SGCs and promoted their proliferation. proBDNF induced apoptosis in SGCs and sensory neurons in DRG following SNT. The proBDNF signaling pathway is a potential novel therapeutic target for reducing sensory neuron and SGCs loss following peripheral nerve injury.

Human adipose-derived stem cells stimulate neuroregeneration.

Traumatic brain injuries and degenerative neurological disorders such as Alzheimer's dementia, Parkinson's disease, amyotrophic lateral sclerosis and many others are characterized by loss of brain cells and supporting structures. Restoring microanatomy and function using stem cells is a promising therapeutic approach. Among the many various sources, adipose-derived stem cells (ADSCs) are one of the most easily harvested alternatives, they multiply rapidly, and they demonstrate low immunogenicity with an ability to differentiate into several cell types. The objective of this study was to evaluate the effect of xenotransplanted human ADSCs on post-traumatic regeneration of rat sciatic nerve. Peripheral reconstruction following complete sciatic transection and autonerve grafting was complemented by intra-operative injection of hADSCs into the proximal and distal stumps. The injury caused gliosis and apoptosis of sensory neurons in the lumbar 5 (L5) ganglia in the control rodents; however, animals treated with hADSCs demonstrated a smaller amount of cellular loss. Formation of amputation neuroma, which hinders axonal repair, was less prominent in the experimental group, and immunohistochemical analysis of myelin basic protein showed good myelination 65days after surgery. At this point, control groups still exhibited high levels of microglia/macrophage-specific marker Iba-1 and proliferating cell nuclear antigen, the mark of an ongoing inflammation and incomplete axonal growth 2months after the injury. This report demonstrates that hADSCs promote neuronal survival in the spinal ganglion, fuel axonal repair and stimulate the regeneration of peripheral nerves.

The Lyme disease spirochete Borrelia burgdorferi induces inflammation and apoptosis in cells from dorsal root ganglia

BackgroundLyme neuroborreliosis (LNB), caused by the spirochete Borrelia burgdorferi, affects both the peripheral and the central nervous systems. Radiculitis or nerve root inflammation, which can cause pain, sensory loss, and weakness, is the most common manifestation of peripheral LNB in humans. We previously reported that rhesus monkeys infected with B. burgdorferi develop radiculitis as well as inflammation in the dorsal root ganglia (DRG), with elevated levels of neuronal and satellite glial cell apoptosis in the DRG. We hypothesized that B. burgdorferi induces inflammatory mediators in glial and neuronal cells and that this inflammatory milieu precipitates glial and neuronal apoptosis.MethodsTo model peripheral neuropathy in LNB we incubated normal rhesus DRG tissue explants with live B. burgdorferi ex vivo and identified immune mediators, producer cells, and verified the presence of B. burgdorferi in tissue sections by immunofluorescence staining and confocal microscopy. We also set up primary cultures of DRG cells from normal adult rhesus macaques and incubated the cultures with live B. burgdorferi. Culture supernatants were subjected to multiplex ELISA to detect immune mediators, while the cells were evaluated for apoptosis by the in situ TUNEL assay. A role for inflammation in mediating apoptosis was assessed by evaluating the above phenomena in the presence and absence of various concentrations of the anti-inflammatory drug dexamethasone. As Schwann cells ensheath the dorsal roots of the DRG, we evaluated the potential of live B. burgdorferi to induce inflammatory mediators in human Schwann cell (HSC) cultures.ResultsRhesus DRG tissue explants exposed to live B. burgdorferi showed localization of CCL2 and IL-6 in sensory neurons, satellite glial cells and Schwann cells while IL-8 was seen in satellite glial cells and Schwann cells. Live B. burgdorferi induced elevated levels of IL-6, IL-8 and CCL2 in HSC and DRG cultures and apoptosis of sensory neurons. Dexamethasone reduced the levels of immune mediators and neuronal apoptosis in a dose dependent manner.ConclusionIn this model, B. burgdorferi induced an inflammatory response and neuronal apoptosis of DRG. These pathophysiological processes could contribute to peripheral neuropathy in LNB.

Intranasal Aspergillus fumigatus exposure induces eosinophilic inflammation and olfactory sensory neuron cell death in mice

To determine the effects of eosinophilic inflammation on olfactory sensory neuron cell death. Mice were sensitized to intranasal Aspergillus fumigatus extract and subsequently challenged acutely or chronically with the same allergen. The olfactory neuroepithelium was assessed for immunohistochemical evidence of apoptosis and inflammation. Sensitized mice challenged with allergen demonstrated elevated eosinophil infiltration of the respiratory and olfactory mucosae, with olfactory sensory neuron apoptosis. Remarkably, massive neuronal apoptosis without eosinophil infiltration occurred in nonsensitized mice after a single dose of extract. Intranasal sensitization with A fumigatus results in a model with multifactorial effects. Protocols using A fumigatus to induce allergic rhinitis may need modification to allow confident interpretation. Fungal allergens may contribute to anosmia through the induction of olfactory sensory neuron apoptosis, with and without prior sensitization.

Brn-3a Transcription Factor Blocks p53-mediated Activation of Proapoptotic Target Genes Noxa and Bax in Vitro and in Vivo to Determine Cell Fate

The Brn-3a POU transcription factor is associated with survival and the differentiation of sensory neuronal cells during development. Brn-3a mediates its effects either by the direct regulation of target genes or indirectly upon interaction with proteins such as p53. Brn-3a differentially regulates p53-mediated gene expression and modifies its effect on cell fate. Here we show that, like Bax, Brn-3a antagonizes p53-mediated transcription of another proapoptotic target, Noxa, significantly reducing transactivation of the Noxa promoter by p53. This effect requires the p53 binding site, and electrophoretic mobility shift assay studies suggest that Brn-3a is associated with p53 when it is bound to its site in the Noxa promoter. The wild type but not the mutant promoter can be immunoprecipitated with Brn-3a in chromatin immunoprecipitation assays. Thus, Brn-3a may act by preventing the recruitment of cofactors required for p53 to transactivate this promoter. The co-expression of Brn-3a and p53 results in decreased endogenous Noxa protein in the neuronal cell line, ND7, suggesting a direct functional effect of this interaction. Moreover, there is a significant elevation of both proapoptotic Bax and Noxa proteins in sensory neuronal tissue taken from Brn-3a-/- embryos during development, compared with wild type controls. Striking changes occurred at embryonic day 14.5, a time that precedes a significant loss of specific neurons in the mutant embryos, but not at embryonic day 16.5 when Brn-3a-expressing cells are already lost by apoptosis. Therefore, the lack of antagonism by Brn-3a on activation of proapoptotic p53 target genes may contribute to the increased apoptosis seen in the Brn-3a-/- embryos. These results support a crucial role for Brn-3a in determining the pathway taken by p53 when co-expressed during development and thus in controlling the fate of these cells.

Differential effects of endogenous brain-derived neurotrophic factor on the survival of axotomized sensory neurons in dorsal root ganglia: A possible role for the p75 neurotrophin receptor

After peripheral nerve injury, axotomized sensory neurons in dorsal root ganglia (DRG) undergo apoptosis and up-regulate brain-derived neurotrophic factor (BDNF). We tested whether endogenous BDNF plays any role in the survival of axotomized sensory neurons using in vitro and in vivo models. In the in vitro model, treatment with BDNF antibody significantly reduced apoptosis of sensory neurons in DRG explants from both adult and neonate rats and adult mice cultured for 48 h. Consistently, exogenous BDNF increased the percentage of apoptotic neurons in the DRGs from mice. The effects of the BDNF antibody and BDNF were not seen in DRGs from p75NTR −/− mice. In the in vivo model, sciatic nerve transection in neonatal rats decreased the total number of neurons in the injured DRG and treatment with antiserum to BDNF significantly exaggerated the loss of DRG neurons. Numbers of sensory neurons expressing BDNF and p75NTR in cultured DRGs increased but that expressing TrkB decreased. In contrast, sciatic nerve transection in vivo reduced the numbers of neurons expressing both p75NTR and TrkB but increased the numbers of cells expressing BDNF, 1 and 7 days after the surgery. These results suggest that BDNF may have differential effects on the survival of sensory neurons depending on the expression of p75NTR. While endogenous BDNF induced apoptosis of axotomized sensory neurons through p75NTR in vitro where more neurons expressed p75NTR, it prevented apoptosis in vivo where fewer neurons expressed p75NTR after sciatic nerve transection.

Treatment of Olfactory Dysfunction, II: Studies With Minocycline

The treatment of anosmia has changed minimally since the early 1970s, despite dramatic advances in the understanding of the molecular biology of olfaction. Recent studies from the authors' laboratory have suggested that most common causes of clinical olfactory dysfunction, including rhinosinusitis, appear to be associated with increased apoptotic death of olfactory sensory neurons. This appears to result in a decline in the number of functioning mature olfactory sensory neurons, despite the capacity of the olfactory epithelium for regeneration. The current study evaluated the ability of the antibiotic minocycline to inhibit olfactory sensory neuron apoptosis. This drug is known to inhibit apoptosis separate from its anti-infective properties. Olfactory sensory neuron apoptosis was triggered by surgical deafferentation ("bulbectomy"), the standard experimental model. Earlier studies have indicated that bulbectomy and sinusitis invoke similar proteolytic enzyme cascades in olfactory sensory neurons. Histological analysis of animal olfactory tissue. Mice underwent unilateral olfactory bulbectomy to induce apoptotic olfactory sensory neuron death, with and without 45 mg/kg intraperitoneal minocycline given 12 hours before surgery and every 12 hours until death. Mice were killed at 2 and 4 days after bulbectomy and assessed for activation of capsase-3 and olfactory sensory neuron survival by immunohistochemical analysis. Minocycline resulted in partial suppression of cell death at 2 days after surgery when compared with untreated animals. Minocycline inhibits olfactory sensory neuron death in the face of a potent pro-apoptotic stimulus. This drug is well tolerated and is currently undergoing human trials for the management of a variety of neurological disorders associated with apoptosis. The current results suggest that minocycline may be efficacious in the management of peripheral olfactory loss as well.

Activation of c-Jun N-terminal kinase mediates gp120IIIB- and nucleoside analogue-induced sensory neuron toxicity

Peripheral neuropathy is the most common neurological symptom in patients with acquired immunodeficiency syndrome (AIDS). Here, we examine possible mechanisms of gp120 and nucleoside reverse transcriptase inhibitors (NRTIs) in the pathogenesis of AIDS peripheral neuropathy. Neonatal dorsal root ganglion (DRG) neurons were found to undergo apoptosis in response to chronic treatment with gp120IIIB, an effect enhanced by the co-application of hCD4, as well as upon exposure to the nucleoside reverse transcriptase inhibitor (NRTI), 2′,3′-dideoxyinosine (ddI). DRG neurons were rescued from the neurotoxic effects of these agents by CEP-1347, an inhibitor of the mixed lineage kinases (MLKs), upstream activators of the c-Jun N-terminal kinase (JNK) signaling pathway. In addition, gp120- or ddI-mediated toxicity were also inhibited by neuronal expression of dominant negative versions of the MLKs. Our results suggest that both gp120 and the NRTIs cause sensory neuron apoptosis through the activation of the JNK pathway, and that CEP-1347-like compounds may serve as a therapeutic option in patients with AIDS-associated peripheral neuropathy.

Activation of c-Jun N-terminal kinase mediates gp120IIIB- and nucleoside analogue-induced sensory neuron toxicity

Peripheral neuropathy is the most common neurological symptom in patients with acquired immunodeficiency syndrome (AIDS). Here, we examine possible mechanisms of gp120 and nucleoside reverse transcriptase inhibitors (NRTIs) in the pathogenesis of AIDS peripheral neuropathy. Neonatal dorsal root ganglion (DRG) neurons were found to undergo apoptosis in response to chronic treatment with gp120IIIB, an effect enhanced by the co-application of hCD4, as well as upon exposure to the nucleoside reverse transcriptase inhibitor (NRTI), 2′,3′-dideoxyinosine (ddI). DRG neurons were rescued from the neurotoxic effects of these agents by CEP-1347, an inhibitor of the mixed lineage kinases (MLKs), upstream activators of the c-Jun N-terminal kinase (JNK) signaling pathway. In addition, gp120- or ddI-mediated toxicity were also inhibited by neuronal expression of dominant negative versions of the MLKs. Our results suggest that both gp120 and the NRTIs cause sensory neuron apoptosis through the activation of the JNK pathway, and that CEP-1347-like compounds may serve as a therapeutic option in patients with AIDS-associated peripheral neuropathy.

Profile of adult rat sensory neuron loss, apoptosis and replacement after sciatic nerve crush

Following permanent transection of the adult rat sciatic nerve, sensory neuron apoptosis in the contributing L4 and L5 dorsal root ganglia can be observed for at least 6 months afterwards. To establish the profile of any sensory neuron apoptosis and loss over time when axonal regeneration is allowed, serial sections of L4 and L5 ganglia were examined and the neurons counted using a stereological technique 1, 2 and 3 months after crushing the right sciatic nerve at mid-thigh level. Our results show that an identical degree of sensory neuron loss and apoptosis occurs 1 month after crush as at 1 month after permanent transection. However, at 3 months no neurons undergoing apoptosis could be observed and no significant loss could be detected in the ipsilateral ganglia when compared to unoperated controls. One explanation was a neuronal replacement mechanism, which was investigated by administering bromodeoxyuridine to rats for 1 month after sciatic nerve transection or crush, prior to detection using immunohistochemistry on sections of their ganglia after 2 months. The presence of bromodeoxyuridine in the nuclei of occasional cells that would be counted as neurons on the basis of size and morphology indicates that a process of apparent neurogenesis may underlie the profile of sensory neuron loss after axotomy.

Susceptibility of sensory neurons to apoptosis following infection by bovine herpesvirus type 1.

Like other members of the alpha subfamily of herpesviruses, bovine herpesvirus type 1 (BHV-1) establishes latent infections in sensory neurons. BHV-1 induces apoptosis in lymphoid cells in vivo and in epithelial cell lines, but the ability of BHV-1 to induce apoptosis in sensory neurons remains unknown. In this report, the susceptibility of rabbit ganglionic neurons to infection by BHV-1 was examined in vitro and in vivo. Following infection of cultured neurons with BHV-1, hallmarks of apoptosis such as chromatin condensation, DNA fragmentation and membrane blebbing were detected. The appearance of these changes was preceded by active viral DNA replication as determined by in situ hybridization. When viral DNA replication was blocked by treatment of cultures with an inhibitor of eukaryotic DNA polymerases, apoptosis but not virus attachment to neurons or bICP0 gene expression was completely prevented. Taken together, these results demonstrate that sensory neurons are not intrinsically resistant to BHV-1-induced apoptosis and that viral DNA replication plays a role in triggering the apoptotic programme. Infection of rabbits with BHV-1 resulted in pathological changes in the trigeminal ganglia (TG) which included mononuclear cell infiltration and neuronophagia. Morphological evidence of apoptosis was not detected in neurons, even in cells with advanced cytophatology. Furthermore, whereas DNA fragmentation was common in infiltrating cells, it was very rare and sporadic in neurons. Therefore, mechanisms in the TG should exist to prevent neuronal apoptosis upon BHV-1 infection.

Target-Independent Specification of Proprioceptive Sensory Neurons

Previous studies in the chick embryo have shown that sensory neurons fail to innervate muscle in the absence of motor neurons. Instead, motor neuron deletion causes more sensory axons to project to the skin. We used this experimental paradigm to determine when sensory neurons are specified to become proprioceptive afferents. Experimental embryos were treated with either saline or exogenous neurotrophin-3 (NT-3) to promote the survival of proprioceptive afferents. In saline-treated embryos, motor neuron deletion caused an increase in sensory neuron apoptosis on the deleted side, an effect reversed by NT3. Motor neuron deletion also eliminated the sartorious muscle nerve, as previously reported. In NT3-treated embryos, this altered nerve pattern was accompanied by the enlargement of the adjacent cutaneous nerve. These embryos were further analyzed by using immunohistochemistry for trkC (a receptor for NT3) retrograde and transganglionic labeling. Our results show that, following motor neuron deletion, more trkC+ afferents project in cutaneous nerves on the deleted side of NT3-treated embryos. Transganglionic labeling demonstrated that at least some of these neurons made spinal projections that are typical of proprioceptive afferents. These results therefore indicate that the proprioceptive phenotype is specified prior to target innervation and that these neurons can retain their identity despite projecting to inappropriate (cutaneous) targets.

Nerve growth factor: structure and function.

Neurotrophins are critical for the development and maintenance of the peripheral and central nervous system. These highly homologous, homodimeric growth factors control cell survival, differentiation, growth cessation, and apoptosis of sensory neurons. The biological functions of the neurotrophins are mediated through two classes of cell surface receptors, the Trk receptors and the p75 neurotrophin receptor (p75NTR). Nerve growth factor (NGF), the best characterized member of the neurotrophin family, sends its survival signals through activation of TrkA and can induce cell death by binding to p75NTR. Recent domain deletion and mutagenesis studies have identified the membrane-proximal domain of the Trks as necessary and sufficient for ligand binding. Crystal structures of this domain of TrkA, TrkB, and TrkC, and an alanine scanning analysis of this domain of TrkA and TrkC have allowed identification of the ligand-binding site. The recent crystal structure of the complex between NGF and the ligand-binding domain of TrkA defines the orientation of NGF in the signaling complex, and eludicates the structural basis for binding and specificity in the family. Further structural work on NGF-TrkA-p7SNTR complexes will be necessary to address the many remaining questions in this complex signaling system.

Apoptosis of sensory neurons and satellite cells after sciatic nerve transection in C57BL/6J mice.

The rate of axonal regeneration, after sciatic nerve lesion in adult C57BL/6J mice, is reduced when compared to other isogenic strains. It was observed that such low regeneration was not due just to a delay, since neuronal death was observed. Two general mechanisms of cell death, apoptosis and necrosis, may be involved. By using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique, we demonstrated that a large number of sensory neurons, as well as satellite cells found in the dorsal root ganglia, were intensely labeled, thus indicating that apoptotic mechanisms were involved in the death process. Although almost no labeled neurons or satellite cells were observed one week after transection, a more than ten-fold increase in TUNEL labeling was detected after two weeks. The results obtained with the C57BL/6J strain were compared with those of the A/J strain, which has a much higher peripheral nerve regeneration potential. In A/J mice, almost no labeling of sensory neurons or satellite cells was observed after one or two weeks, indicating the absence of neuronal loss. Our data confirm previous observations that approximately 40% of C57BL/6J sensory neurons die after sciatic nerve transection, and indicate that apoptotic events are involved. Also, our observations reinforce the hypothesis that the low rate of axonal regeneration occurring in C57BL/6J mice may be the result of a mismatch in the timing of the neurons need for neurotrophic substances, and production of the latter by non-neuronal cells in the distal stump.

Unusual regulation of cyclin D1 and cyclin-dependent kinases cdk2 and cdk4 during in vivo mitotic stimulation of olfactory neuron progenitors in adult mouse.

The molecular mechanisms underlying cell cycle control in neuronal progenitors have been investigated with adult mouse olfactory epithelium as a model system. Odor receptive neurons of mammalian olfactory epithelium are short-lived and renewed in the adult by mitotic division of intrinsic neuronal progenitors. Ablation of the synaptic target, olfactory bulb, induces sequentially extensive apoptosis of sensory neurons and then stimulation of progenitor proliferation, peaking at 36 h and 4 days, respectively, postlesion. Known molecular effectors of G1 phase entry have been assessed on protein extracts of olfactory organs sampled at various postbulbectomy times in adult mice. The decay of betaIII-tubulin and olfactory marker protein levels and the rise of proliferating cell nuclear antigen (PCNA) levels, starting 1 and 3 days, respectively, postlesion, provided the kinetic frame of neuronal dynamics. Cyclin D1, cyclin E, and cyclin-dependent kinase cdk2 levels, low in olfactory organ of intact mice, increased 3 days after bulbectomy in parallel with PCNA levels; cdk4 content was initially high and unaffected by lesioning. Western blots of the known cdk inhibitors revealed proliferation-related decreases of p18, p21, and p27 from high expression in intact organs. Immunoprecipitation of cdk2 and cdk4 fractions of protein extracts at 4 days postlesion (mitotic reaction peak) versus control, followed by cyclin D1 immunoblotting, and vice versa, revealed that levels of both cyclin D1/cdk2 and cyclin D1/cdk4 complexes, as well as their kinase activities, were dramatically increased after lesion. In vivo proliferation of olfactory neuronal lineage cells thus involves functional binding of cyclin D1 with cdk2 and cdk4, with differential activation mechanisms for cdk2 and cdk4. In addition, the RT-PCR-detected cyclin D1 mRNA level remained unaffected after bulbectomy, which indicated that the cyclin D1 rise should involve posttranscriptional mechanisms in this in vivo neuronal system. These observations are discussed, along with their relevance to cell cycle control and to olfactory neuron dynamics.

Inhibition of Sensory Neuron Apoptosis and Prevention of Loss by NT-3 Administration Following Axotomy

Following permanent transection of their peripheral axons, a proportion of adult rat dorsal root ganglion neurons undergo programmed cell death (apoptosis) over a period of months. The underlying causes of this neuron loss are unclear, but may involve the interruption of the supply of target-derived neurotrophic factors, the replacement of which could prevent this loss from occurring. To investigate whether the administration of neurotrophic factors can prevent the dorsal root ganglion neuron death in adults, a 1 mg/ml solution of ciliary neurotrophic factor or of NT-3 was applied via a silicon reservoir to the proximal stump after unilateral sciatic transection at mid-thigh level. The incidence of apoptotic neurons and neuronal loss in the L4 and L5 ganglia ipsilateral to sciatic nerve transection when compared with the contralateral ganglia was then measured 1 month later. This was assessed by examining serial sections of ganglia for neurons undergoing apoptosis and expressing the total counted as a percentage of the total number of neurons estimated using a stereological neuron counting technique. Our results show that NT-3 administration significantly reduced the incidence of apoptotic neurons and prevented neuron loss, while CNTF had no effect on either parameter.