Filip Lim

Regulated Release and Polarized Localization of Brain-Derived Neurotrophic Factor in Hippocampal Neurons

The site and regulation of neurotrophic factor release from neurons is poorly understood. We used a combination of model cell lines and primary culture systems to study the polarity of BDNF sorting and the regulation of its release from hippocampal neurons. Transfection and expression of a human BDNF cDNA in a mouse pituitary cell line, AtT20, resulted in the colocalization of BDNF with the secretory granule marker, chromogranin A. Furthermore, stimulation of these cells with 56 mM KCl or with 5 mM 8-bromo-cAMP increased the release of BDNF approximately 10-to 15-fold within 30 min. To study BDNF release from primary cultures of hippocampal neurons, cells were infected with a defective Herpes Simplex Viral (HSV) vector expressing human BDNF. Depolarizing conditions increased the release of BDNF 5-fold from these cells, further verifying that secretion is regulated. Immunocytochemical analysis using highly specific antibodies determined that endogenous BDNF was predominantly localized to the somatodentritic domain of hippocampal neurons. These findings support the view that BDNF functions as a target-derived signal for afferents to hippocampal pyramidal cells and that it may serve as a regulator of hippocampal plasticity.

FTDP-17 Mutations in tau Transgenic Mice Provoke Lysosomal Abnormalities and Tau Filaments in Forebrain

The tauopathies, which include Alzheimers disease (AD) and frontotemporal dementias, are a group of neurodegenerative disorders characterized by filamentous Tau aggregates. That Tau dysfunction can cause neurodegeneration is indicated by pathogenic tau mutations in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). To investigate how Tau alterations provoke neurodegeneration we generated transgenic mice expressing human Tau with four tubulin-binding repeats (increased by FTDP-17 splice donor mutations) and three FTDP-17 missense mutations: G272V, P301L, and R406W. Ultrastructural analysis of mutant Tau-positive neurons revealed a pretangle appearance, with filaments of Tau and increased numbers of lysosomes displaying aberrant morphology similar to those found in AD. Lysosomal alterations were confirmed by activity analysis of the marker acid phosphatase, which was increased in both transgenic mice and transfected neuroblastoma cells. Our results show that Tau modifications can provoke lysosomal aberrations and suggest that this may be a cause of neurodegeneration in tauopathies.

Chronic lithium treatment decreases mutant tau protein aggregation in a transgenic mouse model.

Tau protein hyperphosphorylation and aggregation into neurofibrillary tangles are characteristic features of several neurodegenerative disorders referred to as tauopathies. Among them, frontotemporal dementia and Parkinsonism linked to chromosome 17 may be caused by dominant missense mutations in the tau gene. Transgenic mice expressing mutant tau serve as valid model systems to study the ethiopathogenesis of these diseases and assay possible therapeutic interventions. Here we report that chronic lithium treatment of a transgenic mouse strain expressing human tau with three missense mutations results in decreased glycogen synthase kinase-3-dependent-tau phosphorylation and a reduction of filamentous aggregates. These data indicate that lithium, presumably acting through the inhibition of glycogen synthase kinase 3, may be useful to curb neurodegeneration in tauopathies.

Accelerated amyloid deposition, neurofibrillary degeneration and neuronal loss in double mutant APP/tau transgenic mice

Even though the idea that amyloid beta peptide accumulation is the primary event in the pathogenesis of Alzheimers disease has become the leading hypothesis, the causal link between aberrant amyloid precursor protein processing and tau alterations in this type of dementia remains controversial. We further investigated the role of beta-amyloid production/deposition in tau pathology and neuronal cell death in the mouse brain by crossing Tg2576 and VLW lines expressing human mutant amyloid precursor protein and human mutant tau, respectively. The resulting double transgenic mice showed enhanced amyloid deposition accompanied by neurofibrillary degeneration and overt neuronal loss in selectively vulnerable brain limbic areas. These findings challenge the idea that tau pathology in Alzheimers disease is merely a downstream effect of amyloid production/deposition and suggest that reciprocal interactions between beta-amyloid and tau alterations may take place in vivo.

The inhibition of phosphatidylinositol-3-kinase induces neurite retraction and activates GSK3

It has been extensively described that neuronal differentiation involves the signalling through neurotrophin receptors to a Ras‐dependent mitogen‐activated protein kinase (MAPK) cascade. However, signalling pathways from other neuritogenic factors have not been well established. It has been reported that cAMP may activate protein kinase (PKA), and it has been shown that PKA‐mediated stimulation of MAPK pathway regulates not only neuritogenesis but also survival. However, extracellular regulated kinases (ERKs) mediated pathways are not sufficient to explain all the processes which occur in neuronal differentiation. Our present data show that: in cAMP‐mediated neuritogenesis, using the SH‐SY5Y human neuroblastoma cell line, there exists a link between the activation of PKA and stimulation of phosphatidylinositol 3‐kinase (PI3K). Both kinase activities are essential to the initial elongation steps. Surprisingly, this neuritogenic process appears to be independent of ERKs. While the activity of PI3K is essential for elongation and maintenance of neurites, its inhibition causes retraction. In this neurite retraction process, GSK3 is activated. Using both a pharmacological approach and gene transfer of a dominant negative form of GSK3, we conclude that this induced retraction is a GSK3‐dependent process which in turn appears to be a common target for transduction pathways involved in lysophosphatidic acid‐mediated and PI3K‐mediated neurite retraction.

Characterization of a double (amyloid precursor protein-tau) transgenic: tau phosphorylation and aggregation.

A double transgenic mouse expressing the amyloid precursor protein, bearing the Swedish mutations, and expressing tau protein containing three of the mutations present in frontotemporal dementia linked to chromosome 17 (FTDP-17), has been characterized. In the double transgenic mouse an increase in tau phosphorylation at serine S262 and S422 was observed compared with that found in simple transgenic mice. The phosphorylation at S262 was also found, in a much lower level, in the single transgenic mouse expressing amyloid precursor protein (APP), and it was absent in that overexpressing tau variant. Additionally, in the double transgenic mouse a slight increase in the amount of sarkosyl insoluble tau polymers was observed in comparison with that found in single transgenic tau mouse. Also, wider tau filaments were found in the double transgenic mouse compared with those found in the single transgenic mouse. Our results suggest that beta-amyloid peptide could facilitate the phosphorylation of tau at a site not directed by proline, such as serine 262, and that modification could facilitate tau aberrant aggregation. Also, they suggest that different types of tau filamentous polymers can occur in different mouse models for tauopathies, like those used for Alzheimers disease or FTDP-17.

The FTDP-17-linked mutation R406W abolishes the interaction of phosphorylated tau with microtubules.

Abstract: The recent finding that several point mutations in the gene encoding for the microtubule‐binding protein tau correlate with neurological disorders has heightened interest in the mechanisms of destabilization of this protein. In this study the functional consequences of the tau mutation R406W on the interaction of the protein with microtubules have been analyzed. Mutated tau is less phosphorylated than its normal counterpart at serines 396 and 404. Furthermore, the phosphorylated mutant protein is unable to bind to microtubules, and, as a consequence, microtubules assembled after transient nocodazole treatment in the presence of this tau variant contain only unmodified tau and appear to form more and longer bundles than those assembled in the presence of wild‐type tau. We propose that phosphorylated tau, unbound to microtubules, could accumulate in the cytoplasm.

Tau function and dysfunction in neurons: its role in neurodegenerative disorders.

Alzheimer’s disease (AD) is the most usual neurodegenerative disorder leading to dementia in the aged human population. It is characterized by the presence of two main brain pathological hallmarks: senile plaques and neurofibrillary tangles (NFTs). NFTs are composed of fibrillar polymers of the abnormally phosphorylated cytoskeletal protein tau.

Glycogen Synthase Kinase-3 Modulates Neurite Outgrowth in Cultured Neurons: Possible Implications for Neurite Pathology in Alzheimer's Disease

Glycogen synthase kinase-3 (GSK-3) is thought to play an important role in the hyperphosphorylation of tau, and possibly other proteins, in Alzheimers disease (AD). However, the effects of GSK-3 on neuronal metabolism are still largely unknown. Here we describe that a low concentration of lithium, which can partially inhibit endogenous GSK-3, favored the extension of neurites from developing neurons, whereas a high concentration of lithium impaired neurite growth. Furthermore, the overexpression of exogenous active GSK-3 in neurons by infection with a defective herpesviral vector blocked neurite growth, which was not affected by either expression of inactive GSK-3 or just the herpesviral vector infection. Neurite extension was restored when neurons overexpressing exogenous active GSK-3 were incubated with lithium. These results are consistent with a role for GSK-3 in the regulation of cytoskeletal dynamics during neurite growth. Accordingly, up-regulation of GSK-3 may contribute to cytoskeletal pathology within neurites in AD.

Infectious Delivery and Expression of a 135 kb Human FRDA Genomic DNA Locus Complements Friedreich's Ataxia Deficiency in Human Cells

Friedreichs ataxia (FA) is the most common recessive ataxia, affecting 1-2 in 50,000 Caucasians, and there is currently no effective cure or treatment. FA results from a deficiency of the mitochondrial protein frataxin brought about by a repeat expansion in intron 1 of the FRDA gene. The main areas affected are the central nervous system (particularly the spinocerebellar system) and cardiac tissue. Therapies aimed at alleviating the neurological degeneration have proved unsuccessful to date. Here, we describe the construction and delivery of high capacity herpes simplex virus type 1 (HSV-1) amplicon vectors expressing the entire 80 kb FRDA genomic locus, driven by the endogenous FRDA promoter and including all introns and flanking regulatory sequences within a 135 kb genomic DNA insert. FA patient primary fibroblasts deficient in frataxin protein and exhibiting sensitivity to oxidative stress were transduced at high efficiency by FRDA genomic locus vectors. Following vector transduction, expression of FRDA protein by immunofluorescence was shown. Finally, functional complementation studies demonstrated restoration of the wild-type cellular phenotype in response to oxidative stress in transduced FA patient cells. These results suggest the potential of the infectious bacterial artificial chromosome-FRDA vectors for gene therapy of FA.