Joost Verhaagen

Brain-derived neurotrophic factor in the ventral midbrain–nucleus accumbens pathway: a role in depression

BACKGROUND Previous work has shown that brain-derived neurotrophic factor (BDNF) and its receptor, tyrosine kinase receptor B (TrkB), are involved in appetitive behavior. Here we show that BDNF in the ventral tegmental area-nucleus accumbens (VTA-NAc) pathway is also involved in the development of a depression-like phenotype. METHODS Brain-derived neurotrophic factor signaling in the VTA-NAc pathway was altered in two complementary ways. One group of rats received intra-VTA infusion of vehicle or BDNF for 1 week. A second group of rats received intra-NAc injections of vehicle or adeno-associated viral vectors encoding full-length (TrkB.FL) or truncated (TrkB.T1) TrkB; the latter is kinase deficient and serves as a dominant-negative receptor. Rats were examined in the forced swim test and other behavioral tests. RESULTS Intra-VTA infusions of BDNF resulted in 57% shorter latency to immobility relative to control animals, a depression-like effect. Intra-NAc injections of TrkB.T1 resulted in and almost fivefold longer latency to immobility relative to TrkB.FL and control animals, an antidepressant-like effect. No effect on anxiety-like behaviors or locomotion was seen. CONCLUSIONS These data suggest that BDNF action in the VTA-NAc pathway might be related to development of a depression-like phenotype. This interpretation is intriguing in that it suggests a role for BDNF in the VTA-NAc that is opposite of the proposed role for BDNF in the hippocampus.

Expression of the gene encoding the chemorepellent semaphorin III is induced in the fibroblast component of neural scar tissue formed following injuries of adult but not neonatal CNS

This study evaluates the expression of the chemorepellent semaphorin III (D)/collapsin-1 (sema III) following lesions to the rat CNS. Scar tissue, formed after penetrating injuries to the lateral olfactory tract (LOT), cortex, perforant pathway, and spinal cord, contained numerous spindle-shaped cells expressing high levels of sema III mRNA. The properties of these cells were investigated in detail in the lesioned LOT. Most sema III mRNA-positive cells were located in the core of the scar and expressed proteins characteristic for fibroblast-like cells. Neuropilin-1, a sema III receptor, was expressed in injured neurons with projections to the lesion site, in a subpopulation of scar-associated cells and in blood vessels around the scar. In contrast to lesions made in the mature CNS, LOT transection in neonates did not induce sema III mRNA expression within cells in the lesion and was followed by vigorous axonal regeneration. The concomitant expression of sema III and its receptor neuropilin-1 in the scar suggests that sema III/neuropilin-1-mediated mechanisms are involved in CNS scar formation. The expression of the secreted chemorepellent sema III following CNS injury provides the first evidence that chemorepulsive semaphorins may contribute to the inhibitory effects exerted by scars on the outgrowth of injured CNS neurites. The vigorous regrowth of injured axons in the absence of sema III following early neonatal lesions is consistent with this notion. The inactivation of sema III in scar tissue by either antibody perturbation or by genetic or pharmacological intervention could be a powerful means to promote long-distance regeneration in the adult CNS.

Injury-induced class 3 semaphorin expression in the rat spinal cord

In this study we evaluate the expression of all members of the class 3 semaphorins and their receptor components following complete transection and contusion lesions of the adult rat spinal cord. Following both types of lesions the expression of all class 3 semaphorins is induced in fibroblast in the neural scar. The distribution of semaphorin-positive fibroblasts differs markedly in scars formed after transection or contusion lesion. In contusion lesions semaphorin expression is restricted to fibroblasts of the meningeal sheet surrounding the lesion, while after transection semaphorin-positive fibroblast penetrate deep into the center of the lesion. Two major descending spinal cord motor pathways, the cortico- and rubrospinal tract, continue to express receptor components for class 3 semaphorins following injury, rendering them potentially sensitive to scar-derived semaphorins. In line with this we observed that most descending spinal cord fibers were not able to penetrate the semaphorin positive portion of the neural scar formed at the lesion site. These results suggest that the full range of secreted semaphorins contributes to the inhibitory nature of the neural scar and thereby may inhibit successful regeneration in the injured spinal cord. Future studies will focus on the neutralization of class 3 semaphorins, in order to reveal whether this creates a more permissive environment for regeneration of injured spinal cord axons.

Alteration of the microRNA network during the progression of Alzheimer's disease

An overview of miRNAs altered in Alzheimers disease (AD) was established by profiling the hippocampus of a cohort of 41 late‐onset AD (LOAD) patients and 23 controls, showing deregulation of 35 miRNAs. Profiling of miRNAs in the prefrontal cortex of a second independent cohort of 49 patients grouped by Braak stages revealed 41 deregulated miRNAs. We focused on miR‐132‐3p which is strongly altered in both brain areas. Downregulation of this miRNA occurs already at Braak stages III and IV, before loss of neuron‐specific miRNAs. Next‐generation sequencing confirmed a strong decrease of miR‐132‐3p and of three family‐related miRNAs encoded by the same miRNA cluster on chromosome 17. Deregulation of miR‐132‐3p in AD brain appears to occur mainly in neurons displaying Tau hyper‐phosphorylation. We provide evidence that miR‐132‐3p may contribute to disease progression through aberrant regulation of mRNA targets in the Tau network. The transcription factor (TF) FOXO1a appears to be a key target of miR‐132‐3p in this pathway.

AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells.

We compared the effects of intravitreal injection of bi-cistronic adeno-associated viral (AAV-2) vectors encoding enhanced green fluorescent protein (GFP) and either ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43) on adult retinal ganglion cell (RGC) survival and regeneration following (i) optic nerve (ON) crush or (ii) after ON cut and attachment of a peripheral nerve (PN). At 7 weeks after ON crush, quantification of βIII-tubulin immunostaining revealed that, compared to AAV-GFP controls, RGC survival was not enhanced by AAV-GAP43-GFP but was increased in AAV-CNTF-GFP (mean RGCs/retina: 17 450±358 s.e.m.) and AAV-BDNF-GFP injected eyes (10 200±4064 RGCs/retina). Consistent with increased RGC viability in AAV-CNTF-GFP and AAV-BDNF-GFP injected eyes, these animals possessed many βIII-tubulin- and GFP-positive fibres proximal to the ON crush. However, only in the AAV-CNTF-GFP group were regenerating RGC axons seen in distal ON (1135±367 axons/nerve, 0.5 mm post-crush), some reaching the optic chiasm. RGCs were immunoreactive for CNTF and quantitative RT-PCR revealed a substantial increase in CNTF mRNA expression in retinas transduced with AAV-CNTF-GFP. The combination of AAV-CNTF-GFP transduction of RGCs with autologous PN-ON transplantation resulted in even greater RGC survival and regeneration. At 7 weeks after PN transplantation there were 27 954 (±2833) surviving RGCs/retina, about 25% of the adult RGC population. Of these, 13 352 (±1868) RGCs/retina were retrogradely labelled after fluorogold injections into PN grafts. In summary, AAV-mediated expression of CNTF promotes long-term survival and regeneration of injured adult RGCs, effects that are substantially enhanced by combining gene and cell-based therapies/interventions.

Viral vectors, tools for gene transfer in the nervous system

Viral vectors are becoming increasingly important tools to investigate the function of neural proteins and to explore the feasibility of gene therapy to treat diseases of the nervous system. This gene transfer technology is based on the use of a virus as a gene delivery vehicle. In contrast to functional analysis of gene products in transgenic mouse, viral vectors can be applied to transfer genes to somatic, post-mitotic cells of fully developed animals. To date, five viral vector systems are available for gene transfer in the nervous system. These include recombinant and defective herpes viral vectors, adenoviral vectors, adeno-associated viral vectors and lentiviral vectors. Of these vectors herpes and adenoviral vectors are the most common in use. To date, one of the main hurdles in applying these two vector systems is the focal immune response that occurs following intraparenchymal infusion. Despite this limitation, herpes and adenoviral vectors have been used successfully to modify the physiological response to injury in several rodent models of neurodegeneration. The first purpose of this review is to describe the principles of the generation of viral vectors and to discuss the advantages and disadvantages of the viral vector systems currently in use for gene transfer in the nervous system. Secondly, we give an overview of the performance of these vectors following direct infusion in the nervous system and review the results obtained with these vectors in animal models of neurodegeneration and regeneration. The results of these initial studies have provided a framework for future experiments based on gene transfer strategies with viral vectors to study normal physiology and pathology of the nervous system.

Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system.

Recombinant adeno-associated virus (rAAV) vectors have become attractive tools for in vivo gene transfer. The production and purification of high-titer rAAV vector stocks for experimental and therapeutic gene transfer continue to undergo improvement. Standard rAAV vector purification protocols include the purification of the vector by cesium chloride (CsCl)-density gradient centrifugation followed by extensive desalination via dialysis against a physiological buffer for in vivo use. These procedures are extremely time consuming and frequently result in a substantial loss of the infectious vector titer. As an alternative to CsCl we have investigated the use of Iodixanol, an X-ray contrast solution, as the density-gradient medium. Purification of rAAV vectors by Iodixanol shortened the centrifugation period to 3 hr and resulted in reproducible concentration and purification of rAAV-vector stocks. We show that injection of rAAV derived from an Iodixanol gradient can be used for in vivo gene transfer applications in the brain and spinal cord without detectable cytopathic effects and directing stable transgene expression for at least 2 months.

Peripheral nerve injury fails to induce growth of lesioned ascending dorsal column axons into spinal cord scar tissue expressing the axon repellent Semaphorin3A

We have investigated the hypothesis that the chemorepellent Semaphorin3A may be involved in the failure of axonal regeneration after injury to the ascending dorsal columns of adult rats. Following transection of the thoracic dorsal columns, fibroblasts in the dorsolateral parts of the lesion site showed robust expression of Semaphorin3A mRNA. In addition, dorsal root ganglion (DRG) neurons with projections through the dorsal columns to the injury site persistently expressed both Semaphorin3A receptor components, neuropilin‐1 and plexin‐A1. These ascending DRG collaterals failed to invade scar regions occupied by Semaphorin3A‐positive fibroblasts, even in animals which had received conditioning lesions of the sciatic nerve to enhance regeneration. Other axon populations in the dorsal spinal cord were similarly unable to penetrate Semaphorin3A‐positive scar tissue. These data suggest that Semaphorin3A may create an exclusion zone for regenerating dorsal column fibres and that enhancing the intrinsic regenerative response of DRG neurons has only limited effects on axonal regrowth. Tenascin‐C and chondroitin sulphate proteoglycans were also detected at the injury site, which was largely devoid of central nervous system (CNS) myelin, showing that several classes of inhibitory factors, including semaphorins, with only partially overlapping spatial and temporal patterns of expression are in a position to participate in preventing regenerative axonal growth in the injured dorsal columns. Interestingly, conditioning nerve injuries enabled numerous ascending DRG axons to regrow across areas of strong tenascin‐C and chondroitin sulphate proteoglycan expression, while areas containing Semaphorin3A and CNS myelin were selectively avoided by (pre)primed axonal sprouts.

Co-localization of high-affinity neurotrophin receptors in nucleus basalis of Meynert neurons and their differential reduction in Alzheimer's disease

It has been suggested that degeneration of neurons in Alzheimers disease is the result of diminished trophic support. However, so far no evidence has been forwarded that neuronal degeneration in Alzheimers disease is causally related to insufficient production of neurotrophins. The present study deals with (i) the expression and co-localization of tyrosine kinase receptors (trks) in the human nucleus basalis of Meynert and (ii) alterations of these receptors in Alzheimers disease in the nucleus basalis of Meynert, an area severely affected in Alzheimers disease. The expression of trkA, trkB and trkC in the nucleus basalis of Meynert of control and Alzheimers disease brains was studied using three polyclonal antibodies specifically recognizing the extracellular domain of trkA, trkB and trkC. Brain material of eight controls and seven Alzheimers disease patients was obtained at autopsy, embedded in paraffin and stained immunocytochemically. Using an image analysis system, we determined the proportion of trk neurons expressing the different trk receptors in controls and Alzheimers disease patients. In control brains, trkA, trkB and trkC were differentially expressed in numerous nucleus basalis of Meynert neurons. The highest proportion of neurons was found to express trkB (75%), followed by trkC (58%) and trkA (54%). Furthermore, using consecutive sections, a clear co-localization of trk receptors was observed in the same neurons. The highest degree of co-localization was observed between trkA and trkB. In Alzheimers disease patients, the number of immunoreactive neurons and the staining intensity of individual neurons was reduced dramatically. Reduction in the proportion of neurons expressing trkA was 69%, in trkB 47% and in trkC 49%, which indicated a differential reduction in the amount of trk receptors in Alzheimers disease. These observations indicate that nucleus basalis of Meynert neurons can be supported by more than one neurotrophin and that the degeneration of these neurons in Alzheimers disease is associated with a decreased expression of trk receptors, suggesting a decreased neurotrophin responsiveness of nucleus basalis of Meynert neurons in Alzheimers disease.

Concerted changes in transcripts in the prefrontal cortex precede neuropathology in Alzheimer's disease.

Using the Braak staging for neurofibrillary changes as an objective indicator of the progression of Alzheimers disease, we have performed a systematic search for global gene expression changes in the prefrontal cortex during the course of Alzheimers disease. In the prefrontal cortex, senile plaques and neurofibrillary changes start to appear around Braak stage III, allowing for the detection of changes in gene expression before, during and after the onset of Alzheimers disease neuropathology. Two distinct patterns of tightly co-regulated groups of genes were observed: (i) an increase in expression in early Braak stages, followed by a decline in expression in later stages (the UPDOWN clusters; containing 865 genes) and (ii) a decrease in expression in early Braak stages, followed by an increase in expression in later stages (the DOWNUP clusters; containing 983 genes). The most profound changes in gene expression were detected between Braak stages II and III, just before or at the onset of plaque pathology and neurofibrillary changes in the prefrontal cortex. We also observed an increase in intracellular beta amyloid staining from Braak stages I to III and a clear decrease in Braak stages IV to VI. These data suggest a link between specific gene expression clusters and Alzheimers disease-associated neuropathology in the prefrontal cortex. Gene ontology over-representation and functional gene network analyses indicate an increase in synaptic activity and changes in plasticity during the very early pre-symptomatic stage of the disease. In later Braak stages, the decreased expression of these genes suggests a reduction in synaptic activity that coincides with the appearance of plaque pathology and neurofibrillary changes and the clinical diagnosis of mild cognitive impairment. The interaction of the ApoE genotype with the expression levels of the genes in the UPDOWN and DOWNUP clusters demonstrates that the accelerating role of ApoE-ε4 in the progression of Alzheimers disease is reflected in the temporal changes in gene expression presented here. Since the UPDOWN cluster contains several genes involved in amyloid precursor protein processing and beta amyloid clearance that increase in expression in parallel with increased intracellular beta amyloid load, just before the onset of plaque pathology in the prefrontal cortex, we hypothesize that the temporally orchestrated increase in genes involved in synaptic activity represents a coping mechanism against increased soluble beta amyloid levels. As these gene expression changes occur before the appearance of Alzheimers disease-associated neuropathology, they provide an excellent starting point for the identification of new targets for the development of therapeutic strategies aimed at the prevention of Alzheimers disease.