Phenotyping animal models of diabetic neuropathy: a consensus statement of the diabetic neuropathy study group of the EASD (Neurodiab).

NIDDK, JDRF, and the Diabetic Neuropathy Study Group of EASD sponsored a meeting to explore the current status of animal models of diabetic peripheral neuropathy. The goal of the workshop was to develop a set of consensus criteria for the phenotyping of rodent models of diabetic neuropathy. The discussion was divided into five areas: (1) status of commonly used rodent models of diabetes, (2) nerve structure, (3) electrophysiological assessments of nerve function, (4) behavioral assessments of nerve function, and (5) the role of biomarkers in disease phenotyping. Participants discussed the current understanding of each area, gold standards (if applicable) for assessments of function, improvements of existing techniques, and utility of known and exploratory biomarkers. The research opportunities in each area were outlined, providing a possible roadmap for future studies. The meeting concluded with a discussion on the merits and limitations of a unified approach to phenotyping rodent models of diabetic neuropathy and a consensus formed on the definition of the minimum criteria required for establishing the presence of the disease. A neuropathy phenotype in rodents was defined as the presence of statistically different values between diabetic and control animals in 2 of 3 assessments (nocifensive behavior, nerve conduction velocities, or nerve structure). The participants propose that this framework would allow different research groups to compare and share data, with an emphasis on data targeted toward the therapeutic efficacy of drug interventions.

Abnormal Intraepidermal Nerve Fiber Density in Disease: A Scoping Review.

Background: Intraepidermal nerve fiber density (IENFD) has become an important biomarker for neuropathy diagnosis and research. The consequences of reduced IENFD can include sensory dysfunction, pain, and a significant decrease in quality of life. We examined the extent to which IENFD is being used as a tool in human and mouse models and compared the degree of fiber loss between diseases to gain a broader understanding of the existing data collected using this common technique. Methods: We conducted a scoping review of publications that used IENFD as a biomarker in human and non-human research. PubMed was used to identify 1,004 initial articles that were then screened to select articles that met the criteria for inclusion. Criteria were chosen to standardize publications so they could be compared rigorously and included having a control group, measuring IENFD in a distal limb, and using protein gene product 9.5 (PGP9.5). Results: We analyzed 397 articles and collected information related to publication year, the condition studied, and the percent IENFD loss. The analysis revealed that the use of IENFD as a tool has been increasing in both human and non-human research. We found that IENFD loss is prevalent in many diseases, and metabolic or diabetes-related diseases were the most studied conditions in humans and rodents. Our analysis identified 74 human diseases in which IENFD was affected, with 71 reporting IENFD loss and an overall average IENFD change of -47%. We identified 28 mouse and 21 rat conditions, with average IENFD changes of -31.6 % and - 34.7% respectively. Additionally, we present data describing sub-analyses of IENFD loss according to disease characteristics in diabetes and chemotherapy treatments in humans and rodents. Interpretation: Reduced IENFD occurs in a surprising number of human disease conditions. Abnormal IENFD contributes to important complications, including poor cutaneous vascularization, sensory dysfunction, and pain. Our analysis informs future rodent studies so they may better mirror human diseases impacted by reduced IENFD, highlights the breadth of diseases impacted by IENFD loss, and urges exploration of common mechanisms that lead to substantial IENFD loss as a complication in disease.

The use of multiple sclerosis condition-specific measures to inform health policy decision-making: mapping from the MSWS-12 to the EQ-5D.

BACKGROUND: Walking impairment has a major influence on the quality of life of people with multiple sclerosis (MS). The Multiple Sclerosis Walking Scale (MSWS-12) assesses the impact of MS on walking ability from the patient's perspective, but in its current form, is not amenable for use in many policy decision-making settings. OBJECTIVES: Statistical 'mapping' methods were used to convert MSWS-12 scores to EQ-5D health state values. METHODS: The relationship between the measures was estimated using cohort data from people with MS in South West England. Regression analyses were conducted, estimation errors assessed, and predictive performance of the best models tested using longitudinal data. RESULTS: Model performance was in line with that of other mapping studies, with the best-performing models being an ordinary least squares (OLS) model using MSWS-12 item scores, and an OLS model using the total MSWS-12 score and its squared term. CONCLUSIONS: A process has been described whereby data from a patient-reported outcome measure (MSWS-12) can be converted to (EQ-5D) health state values. These values may be used to consider the health-related quality of life of people with MS, to estimate quality adjusted life-years for use in effectiveness and cost-effectiveness analyses, and to inform health policy decisions.

Statistical analysis, trial design and duration in Alzheimer's disease clinical trials: a review.

BACKGROUND: The social and economic burden of Alzheimer's disease (AD) and its increasing prevalence has led to much work on new treatment strategies and clinical trials. The search for surrogate markers of disease progression continues but traditional parallel group trial designs that use well-established, but often insensitive, clinical outcome measures predominate. METHODS: We performed a systematic search across the Cochrane Library and PubMed abstracts published between January 2004 and August 2009. Information regarding the clinical trial methodology, outcome measures, intervention type and primary statistical analysis techniques was extracted and categorized, according to a standard protocol. RESULTS: We identified 149 papers describing results from clinical trials in AD containing sufficient detail for our purposes. The largest proportion (38%) presented results of trials based on tests of cognition as the primary outcome measure. The primary analysis in most papers (85%) was a univariate significance test of a single primary outcome measure. CONCLUSIONS: The majority of trials reported a comparison of baseline and end-point assessment over relatively short patient follow-up periods, using univariate statistical methods to compare differences between intervention and control groups in the primary analysis. There is considerable scope to introduce newer statistical methods and trial designs in treatment evaluations in AD.

Characterisation of glyoxalase I in a streptozocin-induced mouse model of diabetes with painful and insensate neuropathy.

AIMS/HYPOTHESIS: Diabetic peripheral neuropathy (DN) is a common complication of diabetes; however, the mechanisms producing positive or negative symptoms are not well understood. The enzyme glyoxalase I (GLO1) detoxifies reactive dicarbonyls that form AGEs and may affect the way sensory neurons respond to heightened AGE levels in DN. We hypothesised that differential GLO1 levels in sensory neurons may lead to differences in AGE formation and modulate the phenotype of DN. METHODS: Inbred strains of mice were used to assess the variability of Glo1 expression by quantitative RT-PCR. Non-diabetic C57BL/6 mice were used to characterise the distribution of GLO1 in neural tissues by immunofluorescence. Behavioural assessments were conducted in diabetic A/J and C57BL/6 mice to determine mechanical sensitivity, and GLO1 abundance was determined by western blot. RESULTS: GLO1 immunoreactivity was found throughout the nervous system, but selectively in small, unmyelinated peptidergic dorsal root ganglia (DRG) neurons that are involved in pain transmission. GLO1 protein was present at various levels in DRG from different inbred mice strains. Diabetic A/J and C57BL/6 mice, two mouse strains with different levels of GLO1, displayed dramatically different behavioural responses to mechanical stimuli. Diabetic C57BL/6 mice also had a reduced abundance of GLO1 following diabetes induction. CONCLUSIONS/INTERPRETATION: These findings reveal that the abundance of GLO1 varies between different murine strains and within different sensory neuron populations. These differences could lead to different responses of sensory neurons to the toxic effects of hyperglycaemia and reactive dicarbonyls associated with diabetes.

Physiological migration of hematopoietic stem and progenitor cells.

Hematopoietic stem cells (HSCs) reside predominantly in bone marrow, but low numbers of HSCs are also found in peripheral blood. We examined the fate of blood-borne HSCs using genetically marked parabiotic mice, which are surgically conjoined and share a common circulation. Parabionts rapidly established stable, functional cross engraftment of partner-derived HSCs and maintained partner-derived hematopoiesis after surgical separation. Determination of the residence time of injected blood-borne progenitor cells suggests that circulating HSCs/progenitors are cleared quickly from the blood. These data demonstrate that HSCs rapidly and constitutively migrate through the blood and play a physiological role in, at least, the functional reengraftment of unconditioned bone marrow.

Introduction of a neurotrophin-3 transgene into muscle selectively rescues proprioceptive neurons in mice lacking endogenous neurotrophin-3.

To clarify the role of muscle-derived neurotrophin-3 (NT-3) in the development of sensory neurons, we generated transgenic mice selectively overexpressing NT-3 in skeletal muscles under the control of a myogenin promoter (myo-NT-3 mice). The myo-NT-3 transgene was then bred into an NT-3 null mutant (-/-) line to generate myo-NT-3, NT-3(-/-) mice in which NT-3 was expressed in muscles, but not elsewhere. Transient overexpression of NT-3 in developing muscles increased the number of proprioceptive neurons as well as the density of both their central and peripheral projections, resulting in more Ia afferents in spinal cord and more spindles (end organs of Ia afferents) in muscles. NT-3 expression restricted to muscles was sufficient to secure the development of proprioceptive neurons and their central and peripheral projections in myo-NT-3, NT-3(-/-) mice. The loss of nonproprioceptive neurons observed in NT-3(-/-) mice was not reversed by the transgene, suggesting that these neurons are regulated by NT-3 from sources other than muscle. We conclude that target-derived rather than intraganglionic NT-3 is preeminent in supporting the development of proprioceptive neurons. The level of NT-3 in developing muscles may be the principal factor determining the number of proprioceptive neurons in dorsal root ganglions and spindles in skeletal muscles of adults.

IB4-binding DRG neurons switch from NGF to GDNF dependence in early postnatal life.

We have tested the role of glial cell line-derived neurotrophic factor (GDNF) in regulating a group of putatively nociceptive dorsal root ganglion (DRG) neurons that do not express calcitonin gene-related peptide (CGRP) and that downregulate the nerve growth factor (NGF) receptor tyrosine kinase, TrkA, after birth. We show that mRNA and protein for the GDNF receptor tyrosine kinase, Ret, are expressed in the DRG in patterns that differ markedly from those of any of the neurotrophin receptors. Most strikingly, a population of small neurons initiates expression of Ret between embryonic day 15.5 and postnatal day 7.5 and maintains Ret expression into adulthood. These Ret-expressing small neurons are selectively labeled by the lectin IB4 and project to lamina IIi of the dorsal horn. Ret-expressing neurons also express the glycosyl-phosphatidyl inositol-linked (GPI-linked) GDNF binding component GDNFR-alpha and retrogradely transport 125I-GDNF, indicating the presence of a biologically active GDNF receptor complex. In vitro, GDNF supports the survival of small neurons that express Ret and bind IB4 while failing to support the survival of neurons expressing TrkA and CGRP. Together, our findings suggest that IB4-binding neurons switch from dependence on NGF in embryonic life to dependence on GDNF in postnatal life and are likely regulated by GDNF in maturity.

Hematopoietic stem cells: challenges to expectations.

The past year provided a number of challenges to our expectations regarding hematopoietic stem cell (HSC) biology. Evidence has emerged that HSCs arise intraembryonically before they can be detected in the yolk sac. A number of genes that may regulate the formation, self-renewal, or differentiation of HSC have been identified. New markers for purifying HSCs have also been described. Although different groups have attributed different properties to HSCs, it now appears that the differences may be explained by variations in assay conditions rather than by differences in the HSCs themselves. Finally, insights have emerged into the complexity of the regulation of HSC proliferation and adhesion properties.

Neurotrophic modulation of myelinated cutaneous innervation and mechanical sensory loss in diabetic mice.

Human diabetic patients often lose touch and vibratory sensations, but to date, most studies on diabetes-induced sensory nerve degeneration have focused on epidermal C-fibers. Here, we explored the effects of diabetes on cutaneous myelinated fibers in relation to the behavioral responses to tactile stimuli from diabetic mice. Weekly behavioral testing began prior to streptozotocin (STZ) administration and continued until 8 weeks, at which time myelinated fiber innervation was examined in the footpad by immunohistochemistry using antiserum to neurofilament heavy chain (NF-H) and myelin basic protein (MBP). Diabetic mice developed reduced behavioral responses to non-noxious (monofilaments) and noxious (pinprick) stimuli. In addition, diabetic mice displayed a 50% reduction in NF-H-positive myelinated innervation of the dermal footpad compared with non-diabetic mice. To test whether two neurotrophins nerve growth factor (NGF) and/or neurotrophin-3 (NT-3) known to support myelinated cutaneous fibers could influence myelinated innervation, diabetic mice were treated intrathecally for 2 weeks with NGF, NT-3, NGF and NT-3. Neurotrophin-treated mice were then compared with diabetic mice treated with insulin for 2 weeks. NGF and insulin treatment both increased paw withdrawal to mechanical stimulation in diabetic mice, whereas NT-3 or a combination of NGF and NT-3 failed to alter paw withdrawal responses. Surprisingly, all treatments significantly increased myelinated innervation compared with control-treated diabetic mice, demonstrating that myelinated cutaneous fibers damaged by hyperglycemia respond to intrathecal administration of neurotrophins. Moreover, NT-3 treatment increased epidermal Merkel cell numbers associated with nerve fibers, consistent with increased numbers of NT-3-responsive slowly adapting A-fibers. These studies suggest that myelinated fiber loss may contribute as significantly as unmyelinated epidermal loss in diabetic neuropathy, and the contradiction between neurotrophin-induced increases in dermal innervation and behavior emphasizes the need for multiple approaches to accurately assess sensory improvements in diabetic neuropathy.

Treatment of congenital pseudarthrosis of the tibia with the Ilizarov technique. Case report and review of the literature.

Congenital pseudarthrosis of the tibia continues to pose one of the most difficult problems in paediatric orthopaedic surgery. The surgical procedures most used for treating congenital pseudarthrosis of the tibia are intramedullary nailing associated with bone grafting, vascularized fibular graft and the Ilizarov external circular fixator. Even when union is achieved, the residual deformities in the affected limb often result in significant disability. These deformities include leg-length discrepancy, angular tibial deformities, ankle mortise valgus and fibular non-union. The Ilizarov method allows simultaneous excision of the pseudarthrosis site, correction of the deformity and lengthening. However, refractures, ankle joint stiffness, fibular non-union with progressive ankle valgus are frequent sequelae with the Ilizarov technique. The surgeon should know when to abandon reconstructive procedures and create a more functional patient with an amputation. The authors discuss the indications and results of the Ilizarov external fixator in two patients with this complex problem. In addition, a critical review of the current literature is undertaken.

Dorsal Root Ganglia Mitochondrial Biochemical Changes in Non-diabetic and Streptozotocin-Induced Diabetic Mice Fed with a Standard or High-Fat Diet.

BACKGROUND: Mitochondrial dysfunction is purported as a contributory mechanism underlying diabetic neuropathy, but a defined role for damaged mitochondria in diabetic nerves remains unclear, particularly in standard diabetes models. Experiments here used a high-fat diet in attempt to exacerbate the severity of diabetes and expedite the time-course in which mitochondrial dysfunction may occur. We hypothesized a high-fat diet in addition to diabetes would increase stress on sensory neurons and worsen mitochondrial dysfunction. METHODS: Oxidative phosphorylation proteins and proteins associated with mitochondrial function were quantified in lumbar dorsal root ganglia. Comparisons were made between non-diabetic and streptozotocin-induced (STZ) C57Bl/6 mice fed a standard or high-fat diet for 8 weeks. RESULTS: Complex III subunit Core-2 and voltage dependent anion channel were increased (by 36% and 28% respectively, p<0.05) in diabetic mice compared to nondiabetic mice fed the standard diet. There were no differences among groups in UCP2, PGC-1α, PGC-1ß levels or Akt, mTor, or AMPK activation. These data suggest compensatory mitochondrial biogenesis occurs to offset potential mitochondrial dysfunction after 8 weeks of STZ-induced diabetes, but a high-fat diet does not alter these parameters. CONCLUSION: Our results indicate mitochondrial protein changes early in STZ-induced diabetes. Interestingly, a high-fat diet does not appear to affect mitochondrial proteins in either nondiabetic or STZ- diabetic mice.

Preparation and properties of glucagon analogs prepared by semi-synthesis from CNBr-glucagon.

The semi-synthetic approach has been used to obtain new analogs of the peptide hormone glucagon. Using the highly purified 27 amino acid fragment of cyanogen bromide-treated glucagon, we have prepared, by nucleophilic addition to the lactone ring, the following derivatives: CNBr-Gly28-glucagon, CNBr-glucagon hydrazide, CNBr-glucagon n-butylamide and CNBr-glucagon biotinamide. Direct aminolysis of the lactone was successful only with sterically unhindered primary amines. Addition of an amino acid could be accomplished by formation of the peptide hydrazide followed by azide coupling. All these analogs were full agonists with decreased potency relative to the native hormone. Examination of the structure-function relationships of these new C-terminal glucagon derivatives suggests that the hydrophobic side-chain of methionine is important to the binding of glucagon to its receptor and that the C-terminal portion of glucagon is only involved in the binding of the hormone to the receptor and not in the transduction process.

Central or peripheral delivery of an adenosine A1 receptor agonist improves mechanical allodynia in a mouse model of painful diabetic neuropathy.

Diabetic peripheral neuropathy is a common complication of diabetes mellitus, and a significant proportion of individuals suffer debilitating pain that significantly affects their quality of life. Unfortunately, symptomatic treatment options have limited efficacy, and often carry significant risk of systemic adverse effects. Activation of the adenosine A1 receptor (A1R) by the analgesic small molecule adenosine has been shown to have antinociceptive benefits in models of inflammatory and neuropathic pain. The current study used a mouse model of painful diabetic neuropathy to determine the effect of diabetes on endogenous adenosine production, and if central or peripheral delivery of adenosine receptor agonists could alleviate signs of mechanical allodynia in diabetic mice. Diabetes was induced using streptozocin in male A/J mice. Mechanical withdrawal thresholds were measured weekly to characterize neuropathy phenotype. Hydrolysis of AMP into adenosine by ectonucleotidases was determined in the dorsal root ganglia (DRG) and spinal cord at 8 weeks post-induction of diabetes. AMP, adenosine and the specific A1R agonist, N(6)-cyclopentyladenosine (CPA), were administered both centrally (intrathecal) and peripherally (intraplantar) to determine the effect of activation of adenosine receptors on mechanical allodynia in diabetic mice. Eight weeks post-induction, diabetic mice displayed significantly decreased hydrolysis of extracellular AMP in the DRG; at this same time, diabetic mice displayed significantly decreased mechanical withdrawal thresholds compared to nondiabetic controls. Central delivery AMP, adenosine and CPA significantly improved mechanical withdrawal thresholds in diabetic mice. Surprisingly, peripheral delivery of CPA also improved mechanical allodynia in diabetic mice. This study provides new evidence that diabetes significantly affects endogenous AMP hydrolysis, suggesting that altered adenosine production could contribute to the development of painful diabetic neuropathy. Moreover, central and peripheral activation of A1R significantly improved mechanical sensitivity, warranting further investigation into this important antinociceptive pathway as a novel therapeutic option for the treatment of painful diabetic neuropathy.

Decreased potency of glucagon on transformed-induced MDCK cells does not reflect an alteration of adenylate cyclase components.

The selective loss of glucagon sensitivity of transformed MDCK cells can be restored by differentiation inducers, a process which requires RNA and protein synthesis and glycosylation. Although the glucagon dose-response curve of normal MDCK cells resembled that of liver and kidney (Kact = 10 nM), the transformed-induced cells were 10-fold less sensitive to the hormone [activation constant (Kact) = 100 nM]. Additionally, the stimulation of cAMP synthesis by a glucagon fragment (glucagon) in transformed-induced cells was greatly reduced compared to normal cells. The adenylate cyclase regulatory components of transformed-induced MDCK cell membranes seemed unaltered compared to the parental line. Both contained equivalent amounts of cholera and pertussis toxin substrates, and soluble extracts were equally capable of reconstituting isoproterenol responsiveness of S49 cyc- membranes. However, membrane fusion studies demonstrated that the glucagon sensitivity of transformed-induced membranes could not be reconstituted with heterologous membranes. When donor transformed-induced membranes (with inactivated adenylate cyclase) were fused with acceptor HeLa membranes (normally unresponsive to glucagon and prostaglandin E), such hybrids were unresponsive to glucagon, although responsiveness to prostaglandin E was evident. Parallel hybrids with normal MDCK membranes were responsive to both glucagon and prostaglandin E. This difference could not be explained by an inhibitory effect of transformed-induced membranes on receptor-adenylate cyclase coupling under the fusion conditions: the ability of these membranes to serve as an acceptor for the reconstitution of vasoactive intestinal peptide responsiveness was identical to that of normal MDCK cells. The data suggest that the glucagon sensitivity induced in transformed MDCK cells differs significantly from that of the parental line. However, these differences cannot be explained by alterations of transformed-induced membrane components relevant to the coupling of hormone receptors to adenylate cyclase.

Partial purification and characterization of the glucagon receptor.

Specific labeling of liver plasma membrane glucagon receptors has been achieved by the photoincorporation of a 125I-labeled photoderivative of glucagon, NE-4-azidophenylamidinoglucagon. Identification of glucagon receptors was facilitated by irradiating membranes in the presence of excess unlabeled glucagon. Isoelectric focusing of radioiodinated membrane proteins revealed one major band of glucagon displaceable material which had an isoelectric point of 5.85. When this material was isolated and run on SDS-polyacrylamide gels a major labeled band of Mr55000 was obtained which had properties consistent with those of the glucagon receptor. These studies indicate that a purification of the glucagon receptor of greater than 700-fold can be attained through the use of isoelectric focusing and SDS-polyacrylamide electrophoresis.

Neurotrophin receptor mRNA expression defines distinct populations of neurons in rat dorsal root ganglia.

The biological actions of neurotrophins are mediated by specific neurotrophin receptor tyrosine kinases (Trks). A low-affinity nerve growth factor (NGF) receptor, p75, appears to modulate sensitivity to neurotrophins in some neuronal populations. It has been recently demonstrated that genes encoding members of the Trk family are expressed in distinct patterns in the dorsal root ganglia (DRG; Mu et al. [1993] (J. Neurosci. 13:4029- 4041). However, the extent to which different neurotrophin receptor genes are coexpressed by individual DRG neurons is unknown. The question of coexpression is important since the expression of more than one member of the trk family by DRG neurons would suggest the potential for regulation by multiple neurotrophins. To address this question, a combination of isotopic and colorimetric in situ hybridization was performed on rat thoracic DRG using riboprobes specific for trkA, trkB, trkC, and p75. We show here that neurons that express trkA are largely distinct from those that express trkC, although there is a small subpopulation that expresses both of these genes. We also show that there is a distinct population of DRG neurons that expresses trkB and does not coexpress either trkA or trkC. P75 is expressed in almost all neurons that express trkA or trkB, but is coexpressed in only 50% of trkC-expressing neurons. Importantly, p75 is not expressed in DRG neurons independent of trk expression. Finally, a subpopulation of DRG neurons does not express any of the neurotrophin receptor mRNAs. Our results demonstrate that there are distinct populations of DRG neurons that express each member of the neurotrophin receptor tyrosine kinase family. Our findings of extensive colocalization of p75 with trkA and trkB lend support to the idea that p75 is important in mediating the actions of NGF and brain-derived neurotrophic factor on DRG neurons. Interestingly, however, p75 expression is clearly unimportant for a subpopulation of neurons that require neurotrophin-3. The fact that p75 is not expressed in the absence of trkA, trkB, or trkC suggests that the function of p75 is closely related to functions of the known neurotrophin-receptor tyrosine kinases. Finally, our results suggest that a significant percentage of DRG neurons may be regulated by non-neurotrophin neuronal growth factors.

Gonadotropin hormone-releasing hormone (GnRH) immunoreactivity in the mesencephalon of sharks and rays.

Other than association with the terminal nerve (TN), little is known concerning the distribution of gonadotropin hormone-releasing hormone (GnRH) in elasmobranchs. The purpose of this study was to identify GnRH immunoreactivity in the brains of three elasmobranch species with special regard to the mesencephalon. The round stingray (Urolophus halleri), thornback guitarfish (Platyrhinoidis triseriata), and leopard shark (Triakis semifasciatus) were used and immunocytochemistry was performed with antisera to both salmon and mammalian GnRH. A large GnRH-immunoreactive (ir) nucleus extends rostrocaudally for approximately 1.5 mm along and adjacent to the midline of the midbrain near the area of the oculomotor nerve. GnRH-ir fibers surround the nucleus and are found diffusely throughout the mesencephalon; some of the fibers may contact the ventricle. The medulla and spinal cord contain ir fibers that most likely originate from the midbrain nucleus. Mesencephalic GnRH-ir cell groups have been reported in representatives of all vertebrate classes with the exception of agnathans and mammals. Such a well-developed cell group in elasmobranchs may aid in understanding the evolution of GnRH systems with regard to the mesencephalon as well as providing insight to the functional significance of this cell group. Possible homologies to mesencephalic GnRH systems reported in other vertebrates is discussed as well.

Glial cell line-derived neurotrophic factor-responsive and neurotrophin-3-responsive neurons require the cytoskeletal linker protein dystonin for postnatal survival.

We have investigated the fate of different neurotrophin-responsive subpopulations of dorsal root ganglion neurons in dystonia musculorum (dt) mice. These mice have a null mutation in the cytoskeletal linker protein, dystonin. Dystonin is expressed by all sensory neurons and cross links actin filaments, intermediate filaments, and microtubules. The dt mice undergo massive sensory neurodegeneration postnatally and die at around 4 weeks of age. We assessed the surviving and degenerating neuronal populations by comparing the dorsal root ganglion (DRG) neurons and central and peripheral projections in dt mice and wildtype mice. Large, neurofilament-H-positive neurons, many of which are muscle afferents and are neurotrophin-3 (NT-3)-responsive, were severely decreased in number in dt DRGs. The loss of muscle afferents was correlated with a degeneration of muscle spindles in skeletal muscle. Nerve growth factor (NGF)-responsive populations, which were visualized using calcitonin gene-related peptide and p75, appeared qualitatively normal in the lumbar spinal cord, DRG, and hindlimb skin. In contrast, glial cell line-derived neurotrophic factor (GDNF)-responsive populations, which were visualized using the isolectin B-4 and thiamine monophosphatase, were severely diminished in the lumbar spinal cord, DRG, and hindlimb skin. Analysis of NT-3, NGF, and GDNF mRNA levels using semiquantitative reverse transcriptase-polymerase chain reaction revealed normal trophin synthesis in the peripheral targets of dt mice, arguing against decreased trophic synthesis as a possible cause of neuronal degeneration. Thus, the absence of dystonin results in the selective survival of NGF-responsive neurons and the postnatal degeneration of many NT-3- and GDNF-responsive neurons. Our results reveal that the loss of this ubiquitously expressed cytoskeletal linker has diverse effects on sensory subpopulations. Moreover, we show that dystonin is critical for the maintenance of certain DRG neurons, and its function may be related to neurotrophic support.

The guidance molecule semaphorin III is expressed in regions of spinal cord and periphery avoided by growing sensory axons.

The protein collapsin was purified from chick brain on the basis of its ability to inhibit sensory neuron growth cones, implicating this molecule in sensory axon guidance (Luo et al. [1993] Cell 75:217-227). To examine the relationship between collapsin and sensory axon growth, we examined the pattern of mRNA expression of collapsin's mammalian paralogue, Semaphorin III (Sema III), and compared it to dorsal root ganglion (DRG) axon pathways in the developing rat embryo. Centrally, DRG axons enter the spinal cord by embryonic (E) 11 and branch into the gray matter by E15 in brachial and thoracic regions. Laminar specific targets are reached by E17. Between E13 and E17, Sema III mRNA is expressed at high levels in the entire ventral half of the spinal cord except the floor plate. This pattern suggests that Sema III may inhibit non-proprioceptive sensory axons from penetrating the ventral spinal cord. Peripherally, sensory axons have entered the anterior sclerotome by E11 at all rostrocaudal levels. At this age, Sema III mRNA is already expressed in the dermamyotome and ventral aspect of the posterior sclerotome, areas which axons pass between but do not penetrate en route to their peripheral targets. From E12 to E15, the axons lengthen and branch into smaller fascicles which extend toward peripheral targets. During this time, Sema III mRNA is expressed by many mesodermal structures surrounding the axon fascicles, with highest levels observed in the dermamyotome, perinotochordal mesenchyme, pelvic girdle, and limb. As development proceeds, Sema III mRNA expression is quickly downregulated before disappearing by birth. Taken together, our results demonstrate that the gene for Sema III is expressed in central and peripheral regions which are avoided by growing DRG axons. These findings are consistent with the idea that Sema III inhibits growth and branching of axons into inappropriate areas during development.