Gene Therapy Vector Encoding Neuropeptide Y and Its Receptor Y2 for Future Treatment of Epilepsy: Preclinical Data in Rats.

Gene therapy to treat pharmacoresistant temporal lobe epilepsy in humans is now being developed using an AAV vector (CG01) that encodes the combination of neuropeptide Y and its antiepileptic receptor Y2. With this in mind, the present study aimed to provide important preclinical data on the effects of CG01 on the duration of transgene expression, cellular tropism, and potential side effects on body weight and cognitive function. The CG01 vector was administered unilaterally into the dorsal and ventral hippocampus of adult male rats and expression of both transgenes was found to remain elevated without a sign of decline at 6 months post-injection. CG01 appeared to mediate expression selectively in hippocampal neurons, without expression in astrocytes or oligodendrocytes. No effects were seen on body weight as well as on short- or long-term memory as revealed by testing in the Y-maze or Morris water maze tests. Thus these data show that unilateral CG01 vector treatment as future gene therapy in pharmacoresistant temporal lobe epilepsy patients should result in stable and long-term expression predominantly in neurons and be well tolerated without side effects on body weight and cognitive function.

Revisiting the peripheral sink hypothesis: inhibiting BACE1 activity in the periphery does not alter ß-amyloid levels in the CNS.

Aggregation of amyloid beta (Aß) peptides and the subsequent neural plaque formation is a central aspect of Alzheimer's disease. Various strategies to reduce Aß load in the brain are therefore intensely pursued. It has been hypothesized that reducing Aß peptides in the periphery, that is in organs outside the brain, would be a way to diminish Aß levels and plaque load in the brain. In this report, we put this peripheral sink hypothesis to test by investigating how selective inhibition of Aß production in the periphery using a ß-secretase (BACE)1 inhibitor or reduced BACE1 gene dosage affects Aß load in the brain. Selective inhibition of peripheral BACE1 activity in wild-type mice or mice over-expressing amyloid precursor protein (APPswe transgenic mice; Tg2576) reduced Aß levels in the periphery but not in the brain, not even after chronic treatment over several months. In contrast, a BACE1 inhibitor with improved brain disposition reduced Aß levels in both brain and periphery already after acute dosing. Mice heterozygous for BACE1, displayed a 62% reduction in plasma Aß40, whereas brain Aß40 was only lowered by 11%. These data suggest that reduction of Aß in the periphery is not sufficient to reduce brain Aß levels and that BACE1 is not the rate-limiting enzyme for Aß processing in the brain. This provides evidence against the peripheral sink hypothesis and suggests that a decrease in Aß via BACE1 inhibition would need to be carried out in the brain. Aggregation of amyloid beta (Aß) peptides in the brain is a central aspect of Alzheimer's disease. In this study, we demonstrate that inhibition of Aß formation by BACE1 inhibitors needs to be carried out in the brain and that reduction of Aß in the periphery is not sufficient to reduce brain Aß levels. This information is useful for developing future Aß-targeting therapies for Alzheimer's disease.

Elevated MARK2-dependent phosphorylation of Tau in Alzheimer's disease.

The appearance of neurofibrillary tangles (NFT), one of the major hallmarks of Alzheimer's disease (AD), is most likely caused by inappropriate phosphorylation and/or dephosphorylation of tau, eventually leading to the accumulation of NFTs. Enhanced phosphorylation of tau on Ser(262) is detected early in the course of the disease and may have a role in the formation of tangles. Several kinases such as microtubule-affinity regulating kinase (MARK), protein kinase A, calcium calmodulin kinase II, and checkpoint kinase 2 are known to phosphorylate tau on Ser(262) in vitro. In this study, we took advantage of the in situ proximity ligation assay to investigate the role of MARK2, one of the four MARK isoforms, in AD. We demonstrate that MARK2 interacts with tau and phosphorylates tau at Ser(262) in stably transfected NIH/3T3 cells expressing human recombinant tau. Staurosporine, a protein kinase inhibitor, significantly reduced the interaction between MARK2 and tau, and also phosphorylation of tau at Ser(262). Furthermore, we observed elevated interactions between MARK2 and tau in post-mortem human AD brains, compared to samples from non-demented elderly controls. Our results from transfected cells demonstrate a specific interaction between MARK2 and tau, as well as MARK2-dependent phosphorylation of tau at Ser(262). Furthermore, the elevated interactions between MARK2 and tau in AD brain sections suggests that MARK2 may play an important role in early phosphorylation of tau in AD, possibly qualifying as a therapeutic target for intervention to prevent disease progression.

Decreased axonal transport rates in the Tg2576 APP transgenic mouse: improvement with the gamma-secretase inhibitor MRK-560 as detected by manganese-enhanced MRI.

Neuropil deposition of beta-amyloid (Aß) peptides is believed to be a key event in the neurodegenerative process of Alzheimer's disease (AD). An early and consistent clinical finding in AD is olfactory dysfunction with associated pathology. Interestingly, transgenic amyloid precursor protein (Tg2576) mice also show early amyloid pathology in olfactory regions. Moreover, a recent study indicates that axonal transport is compromised in the olfactory system of Tg2576 mice, as measured by manganese-enhanced magnetic resonance imaging (MEMRI). Here we tested whether the putative axonal transport deficit in the Tg2576 mouse model improves in response to a selective gamma-secretase inhibitor, N-[cis-4-[(4-chlorophenyl)-sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560). Tg2576 mice or wild-type (WT) littermates were treated daily with MRK-560 (30 µmol/kg) or vehicle for 4 (acute) or 29 days (chronic). The subsequent MEMRI analysis revealed a distinct axonal transport dysfunction in the Tg2576 mice compared with its littermate controls. Interestingly, the impairment of axonal transport could be fully reversed by chronic administration of MRK-560, in line with the significantly lowered levels of both soluble and insoluble forms of Aß found in the brain and olfactory bulbs (OBs) following treatment. However, no improvement of axonal transport was observed after acute treatment with MRK-560, where soluble but not insoluble forms of Aß were reduced in the brain and OBs. The present results show that axonal transport is impaired in Tg2576 mice compared with WT controls, as measured by MEMRI. Chronic treatment in vivo with a gamma-secretase inhibitor, MRK-560, significantly reduces soluble and insoluble forms of Aß, and fully reverses the axonal transport dysfunction.

Evidence for dimeric BACE-mediated APP processing.

beta-Secretase (BACE) is an aspartyl protease, which proteolytically processes amyloid precursor protein, making BACE an interesting pharmacological target in Alzheimer's disease. To study the enzymatic function of BACE, we mutated either of the two aspartic acid residues in the active site of BACE. This rendered BACE functionally inactive without affecting the degree of glycosylation or endosomal localization. In contrast, substituting both active site aspartic acid residues produced a functionally inactive, endoplasmic reticulum-retained and partially glycosylated BACE. Interestingly, co-expression of the two single active site mutants partially restored beta-site cleavage of amyloid precursor protein, and the restored activity was inhibited with similar dose-dependency and potency as wildtype BACE by a small molecule inhibitor raised against BACE. In sum, our data suggest that two different active site mutants can complement each other in a partially functional BACE dimer and mediate APP processing.

Identification of non-muscle myosin heavy chain as a substrate for Cdk5 and tool for drug screening.

BACKGROUND: Deregulated activation of cyclin-dependent kinase-5 (Cdk5) is implicated in neurodegenerative disorders such as Alzheimer's disease. One of the restricting factors for developing specific Cdk5 inhibitors is the lack of reproducible and well-characterized cellular in vitro assay systems. METHODS: HEK293 cells were transfected with Cdk5 and its activator p25 as a starting point for an assay to screen for Cdk5 kinase inhibitors. To identify suitable substrates for Cdk5 we utilized an antibody that recognizes phospho serine in a consensus motif for Cdk substrates. RESULTS: Western blot analysis of transfected cells detected a 200 kDa band that was identified, by mass spectrometry, as non-muscle myosin heavy chain, type B (NMHC-B). Phosphorylation of NMHC-B was evident only in cells that were double transfected with Cdk5/p25 and was dose-dependently inhibited by Roscovitine and other Cdk5 inhibitors. Cdk5 was found to phosphorylate NMHC-B also in the human neuroblastoma SH-SY5Y cell line. CONCLUSION: A novel Cdk5 substrate NMHC-B was identified in this study. A cellular assay for screening of Cdk5 inhibitors was established using NMHC-B phosphorylation as a read-out in Cdk5/p25 transfected HEK293 cells. A novel Cdk5 inhibitor was also pharmacologically characterized in this assay system.

Brain-derived neurotrophic factor selectively regulates dendritogenesis of parvalbumin-containing interneurons in the main olfactory bulb through the PLCgamma pathway.

Molecular mechanisms of neurotrophin signaling on dendrite development and dynamics are only partly understood. To address the role of brain-derived neurotrophic factor (BDNF) in the morphogenesis of GABAergic neurons of the main olfactory bulb, we analyzed mice lacking BDNF, mice carrying neurotrophin-3 (NT3) in the place of BDNF, and TrkB signaling mutant mice with a receptor that can activate phospholipase Cgamma (PLCgamma) but is unable to recruit the adaptors Shc/Frs2. BDNF deletion yielded a compressed olfactory bulb with a significant loss of parvalbumin (PV) immunoreactivity in GABAergic interneurons of the external plexiform layer. Dendrite development of PV-positive interneurons was selectively attenuated by BDNF since other Ca2+ -binding protein-containing neuron populations appeared unaffected. The deficit in PV-positive neurons could be rescued by the NT3/NT3 alleles. The degree of PV immunoreactivity was dependent on BDNF and TrkB recruitment of the adaptor proteins Shc/Frs2. In contrast, PLCgamma signaling from the TrkB receptor was sufficient for dendrite growth in vivo and consistently, blocking PLCgamma prevented BDNF-dependent dendrite development in vitro. Collectively, our results provide genetic evidence that BDNF and TrkB signaling selectively regulate PV expression and dendrite growth in a subset of neurochemically-defined GABAergic interneurons via activation of the PLCgamma pathway.

Genetic evidence for selective neurotrophin 3 signalling through TrkC but not TrkB in vivo.

Neurotrophins control neuronal survival in a target-derived manner during the period of naturally occurring cell death in development. The specificity of this mechanism has been attributed to a restricted spatio-temporal expression of neurotrophin ligands in target tissues, as well as a selective expression of their cognate tyrosine kinase (Trk) receptors in different neuronal subpopulations. However, several in vitro and in vivo studies of null mutant mice have suggested that neurotrophin 3 (NT 3) also signals through the non-preferred TrkB receptor. In this study, we have directly addressed the in vivo preference of NT 3 to signal through TrkB or TrkC, by crossing the NT 3 knock-in mice (BDNF(NT 3/NT 3) mice) with the TrkB- or TrkC-null mutant mice. We find that TrkB is dispensable, whereas TrkC is required for the neuronal rescue by the NT 3 allele in the brain-derived neurotrophic factor- and NT 3-dependent cochleovestibular system. Our results show that NT 3 maintains survival of cells as well as target innervation only through interactions with TrkC in vivo. TrkB and TrkC receptors are thus not functionally redundant for NT 3, even when coexpressed in neurons of the cochleovestibular system.

Lingual deficits in neurotrophin double knockout mice.

Brain-derived neurotrophic factor (BDNF) and Neurotrophin 3 (NT-3) are members of the neurotrophin family and are expressed in the developing and adult tongue papillae. BDNF null-mutated mice exhibit specific impairments related to innervation and development of the gustatory system while NT-3 null mice have deficits in their lingual somatosensory innervation. To further evaluate the functional specificity of these neurotrophins in the peripheral gustatory system, we generated double BDNF/NT-3 knockout mice and compared the phenotype to BDNF(-/-) and wild-type mice. Taste papillae morphology was severely distorted in BDNF(-/-) xNT-3(-/-) mice compared to single BDNF(-/-) and wild-type mice. The deficits were found throughout the tongue and all gustatory papillae. There was a significant loss of fungiform papillae and the papillae were smaller in size compared to BDNF(-/-) and wild-type mice. Circumvallate papillae in the double knockouts were smaller and did not contain any intraepithelial nerve fibers. BDNF(-/-) xNT-3(-/-) mice exhibited additive losses in both somatosensory and gustatory innervation indicating that BDNF and NT-3 exert specific roles in the innervation of the tongue. However, the additional loss of fungiform papillae and taste buds in BDNF(-/-) xNT-3(-/-) mice compared to single BDNF knockout mice indicate a synergistic functional role for both BDNF-dependent gustatory and NT-3-dependent somatosensory innervations in taste bud and taste papillae innervation and development.

Differential influence of BDNF and NT3 on the expression of calcium binding proteins and neuropeptide Y in vivo.

Neurotrophins enhance and maintain some neuronal phenotypes and suppress others by influencing the expression of neuropeptides and calcium binding proteins, thereby affecting many different physiological functions of the brain. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) show different effects on neuronal phenotypes despite largely overlapping expression of their respective receptors, TrkB and TrkC. Using BDNF null mutant (BDNF-/-) mice and mice where the protein coding DNA sequence of BDNF has been replaced by NT3 (BDNFNT3/NT3 mice), we have analysed the roles of BDNF and NT3 in controlling neuropeptide and calcium binding protein expression in the brain. Our results show that NT3 expressed emporally and spatially in the place of BDNF is sufficient in some neuronal populations to compensate for the loss of BDNF.

BDNF gene replacement reveals multiple mechanisms for establishing neurotrophin specificity during sensory nervous system development.

Neurotrophins have multiple functions during peripheral nervous system development such as controlling neuronal survival, target innervation and synaptogenesis. Neurotrophin specificity has been attributed to the selective expression of the Trk tyrosine kinase receptors in different neuronal subpopulations. However, despite overlapping expression of TrkB and TrkC in many sensory ganglia, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) null mutant mice display selective losses in neuronal subpopulations. In the present study we have replaced the coding part of the BDNF gene in mice with that of NT3 (BDNF(NT3/NT3)) to analyse the specificity and selective roles of BDNF and NT3 during development. Analysis of BDNF(NT3/NT3) mice showed striking differences in the ability of NT3 to promote survival, short-range innervation and synaptogenesis in different sensory systems. In the cochlea, specificity is achieved by a tightly controlled spatial and temporal ligand expression. In the vestibular system TrkB or TrkC activation is sufficient to promote vestibular ganglion neuron survival, while TrkB activation is required to promote proper innervation and synaptogenesis. In the gustatory system, NT3 is unable to replace the actions of BDNF possibly because of a temporally selective expression of TrkB in taste neurons. We conclude that there is no general mechanism by which neurotrophin specificity is attained and that specificity is achieved by (i) a tightly controlled spatial and temporal expression of ligands, (ii) different Trk receptors playing distinct roles within the same neuronal subpopulation, or (iii) selective receptor expression in sensory neuron subpopulations.

Activation of the TrkB neurotrophin receptor is induced by antidepressant drugs and is required for antidepressant-induced behavioral effects.

Recent studies have indicated that exogenously administered neurotrophins produce antidepressant-like behavioral effects. We have here investigated the role of endogenous brain-derived neurotrophic factor (BDNF) and its receptor trkB in the mechanism of action of antidepressant drugs. We found that trkB.T1-overexpressing transgenic mice, which show reduced trkB activation in brain, as well as heterozygous BDNF null (BDNF(+/)-) mice, were resistant to the effects of antidepressants in the forced swim test, indicating that normal trkB signaling is required for the behavioral effects typically produced by antidepressants. In contrast, neurotrophin-3(+/)- mice showed a normal behavioral response to antidepressants. Furthermore, acute as well as chronic antidepressant treatment induced autophosphorylation and activation of trkB in cerebral cortex, particularly in the prefrontal and anterior cingulate cortex and hippocampus. Tyrosines in the trkB autophosphorylation site were phosphorylated in response to antidepressants, but phosphorylation of the shc binding site was not observed. Nevertheless, phosphorylation of cAMP response element-binding protein was increased by antidepressants in the prefrontal cortex concomitantly with trkB phosphorylation and this response was reduced in trkB.T1-overexpressing mice. Our data suggest that antidepressants acutely increase trkB signaling in a BDNF-dependent manner in cerebral cortex and that this signaling is required for the behavioral effects typical of antidepressant drugs. Neurotrophin signaling increased by antidepressants may induce formation and stabilization of synaptic connectivity, which gradually leads to the clinical antidepressive effects and mood recovery.

Pharmacological strategies for preventing cochlear damage induced by noise trauma.

Hearing loss induced by noise, as well as in combination with other environmental factors, is a significant health problem throughout the world. Although most structures in the inner ear can be harmed by excessive sound exposure, the sensory cells are the most vulnerable. Damage to the stereocilia bundle is often the first structural alteration noted. Once a large number of hair cells are lost, the nerve fibres to that region also degenerate resulting in an irreversible hearing loss. At present, the underlying mechanism for cochlear damage induced by noise is not fully understood. The failure of the adult peripheral auditory system to regenerate after injury is a major clinical problem. However, a number of experimental applications have recently become available and are effective in reducing the damaging effects of noise. Current experimental designs include strategies for protecting against injury and are primarily based on the fact that the metabolic state of the cochlea can determine the overall degree of hearing loss induced by noise. The purpose of the present article is to review the current literature dealing with strategies for protecting against noise trauma.