Ex vivo adenoviral vector-mediated neurotrophin gene transfer to olfactory ensheathing glia: Effects on rubrospinal tract regeneration, lesion size, and functional recovery after implantation in the injured rat spinal cord

The present study uniquely combines olfactory ensheathing glia (OEG) implantation with ex vivo adenoviral (AdV) vector-based neurotrophin gene therapy in an attempt to enhance regeneration after cervical spinal cord injury. Primary OEG were transduced with AdV vectors encoding rat brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or bacterial marker protein -galactosidase (LacZ) and subsequently implanted into adult Fischer rats directly after unilateral transection of the dorsolateral funiculus. Implanted animals received a total of 2 x 105 OEG that were subjected to transduction with neurotrophin-encoding AdV vector, AdV-LacZ, or no vector, respectively. At 4 months after injury, lesion volumes were smaller in all OEG implanted rats and significantly reduced in size after implantation of neurotrophin-encoding AdV vector-transduced OEG. All OEG grafts were filled with neurofilament-positive axons, and AdV vector-mediated expression of BDNF by implanted cells significantly enhanced regenerative sprouting of the rubrospinal tract. Behavioral analysis revealed that OEG-implanted rats displayed better locomotion during horizontal rope walking than unimplanted lesioned controls. Recovery of hind limb function was also improved after implantation of OEG that were transduced with a BDNF- or NT-3-encoding AdV vector. Hind limb performance during horizontal rope locomotion did directly correlate with lesion size, suggesting that neuroprotective effects of OEG implants contributed to the level of functional recovery. Thus, our results demonstrate that genetic engineering of OEG not only resulted in a cell that was more effective in promoting axonal outgrowth but could also lead to enhanced recovery after injury, possibly by sparing of spinal tissue

PRRT2-related phenotypes in patients with a 16p11.2 deletion

We studied the presence of benign infantile epilepsy (BIE), paroxysmal kinesigenic dyskinesia (PKD), and PKD with infantile convulsions (PKD/IC) in patients with a 16p11.2 deletion including PRRT2 or with a PRRT2 loss-of-function sequence variant. Index patients were recruited from seven Dutch university hospitals. The presence of BIE, PKD and PKD/IC was retrospectively evaluated using questionnaires and medical records. We included 33 patients with a 16p11.2 deletion: three (9%) had BIE, none had PKD or PKD/IC. Twelve patients had a PRRT2 sequence variant: BIE was present in four (p = 0.069), PKD in six (p < 0.001) and PKD/IC in two (p = 0.067). Most patients with a deletion had undergone genetic testing because of developmental problems (87%), whereas all patients with a sequence variant were tested because of a movement disorder (55%) or epilepsy (45%). BIE, PKD and PKD/IC clearly showed incomplete penetrance in patients with 16p11.2 deletions, but were found in all and 95% of patients with a PRRT2 sequence variant in our study and a large literature cohort, respectively. Deletions and sequence variants have the same underlying loss-of-function disease mechanism. Thus, differences in ascertainment have led to overestimating the frequency of BIE, PKD and PKD/IC in patients with a PRRT2 sequence variant. This has important implications for counseling if genome-wide sequencing shows such variants in patients not presenting the PRRT2-related phenotypes

Colocalization 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 Alzheimer's disease is the result of diminished trophic support. However, so far no evidence has been forwarded that neuronal degeneration in Alzheimer's 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 Alzheimer's disease in the nucleus basalis of Meynert, an area severely affected in Alzheimer's disease. The expression of trkA, trkB and trkC in the nucleus basalis of Meynert of control and Alzheimer's 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 Alzheimer's 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 Alzheimer's 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 Alzheimer's 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 Alzheimer's 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 Alzheimer's disease is associated with a decreased expression of trk receptors, suggesting a decreased neurotrophin responsiveness of nucleus basalis of Meynert neurons in Alzheimer's diseas

The use of adenoviral vectors and ex vivo transduced neurotransplants: towards promotion of neuroregeneration

Regeneration of injured axons following injury depends on a delicate balance between growth-promoting and growth-inhibiting factors. Overexpression of neurotrophin genes seems a promising strategy to promote regeneration. Trophic genes can be overexpressed at the site of injury at the axonal stumps, or at the perikaryal level of the injured neuron. Transduction of the neural cells can be achieved by applying adenoviral vectors, either directly in vivo or-in the case of neurotransplantation as an ex vivo approach. In both cases it would create a more permissive environment for axonal growth and therefore in functional regeneration. In this article, the feasibility of the use of adenoviral vectors in several neuroregeneration models--in particularly in spinal cord lesion models and the biological clock transplantation model--is illustrated. The results show that the adenoviral vectors can be a powerful tool to study the effects of overexpression of genes in an in vivo paradigm of nerve regeneration or nerve outgrowth. The potential use of adenoviral vectors and ex vivo transduced neurotransplants is discussed

Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks of 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