Kurt Spittaels

Prominent Axonopathy in the Brain and Spinal Cord of Transgenic Mice Overexpressing Four-Repeat Human tau Protein

Mutations in the human tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17. Some mutations, including mutations in intron 10, induce increased levels of the functionally normal four-repeat tau protein isoform, leading to neurodegeneration. We generated transgenic mice that overexpress the four-repeat human tau protein isoform specifically in neurons. The transgenic mice developed axonal degeneration in brain and spinal cord. In the model, axonal dilations with accumulation of neurofilaments, mitochondria, and vesicles were documented. The axonopathy and the accompanying dysfunctional sensorimotor capacities were transgene-dosage related. These findings proved that merely increasing the concentration of the four-repeat tau protein isoform is sufficient to injure neurons in the central nervous system, without formation of intraneuronal neurofibrillary tangles. Evidence for astrogliosis and ubiquitination of accumulated proteins in the dilated part of the axon supported this conclusion. This transgenic model, overexpressing the longest isoform of human tau protein, recapitulates features of known neurodegenerative diseases, including Alzheimers disease and other tauopathies. The model makes it possible to study the interaction with additional factors, to be incorporated genetically, or with other biological triggers that are implicated in neurodegeneration.

Glycogen Synthase Kinase-3β Phosphorylates Protein Tau and Rescues the Axonopathy in the Central Nervous System of Human Four-repeat Tau Transgenic Mice

Protein tau filaments in brain of patients suffering from Alzheimers disease, frontotemporal dementia, and other tauopathies consist of protein tau that is hyperphosphorylated. The responsible kinases operating in vivo in neurons still need to be identified. Here we demonstrate that glycogen synthase kinase-3β (GSK-3β) is an effective kinase for protein tau in cerebral neurons in vivo in adult GSK-3β and GSK-3β × human tau40 transgenic mice. Phosphorylated protein tau migrates slower during electrophoretic separation and is revealed by phosphorylation-dependent anti-tau antibodies in Western blot analysis. In addition, its capacity to bind to re-assembled paclitaxel (Taxol®)-stabilized microtubules is reduced, compared with protein tau isolated from mice not overexpressing GSK-3β. Co-expression of GSK-3β reduces the number of axonal dilations and alleviates the motoric impairment that was typical for single htau40 transgenic animals (Spittaels, K., Van den Haute, C., Van Dorpe, J., Bruynseels, K., Vandezande, K., Laenen, I., Geerts, H., Mercken, M., Sciot, R., Van Lommel, A., Loos, R., and Van Leuven, F. (1999) Am. J. Pathol. 155, 2153–2165). Although more hyperphosphorylated protein tau is available, neither an increase in insoluble protein tau aggregates nor the presence of paired helical filaments or tangles was observed. These findings could have therapeutic implications in the field of neurodegeneration, as discussed.

Neuronal Deficiency of Presenilin 1 Inhibits Amyloid Plaque Formation and Corrects Hippocampal Long-Term Potentiation But Not a Cognitive Defect of Amyloid Precursor Protein [V717I] Transgenic Mice

In the brain of Alzheimers disease (AD) patients, neurotoxic amyloid peptides accumulate and are deposited as senile plaques. A major therapeutic strategy aims to decrease production of amyloid peptides by inhibition of γ-secretase. Presenilins are polytopic transmembrane proteins that are essential for γ-secretase activity during development and in amyloid production. By loxP/Cre-recombinase-mediated deletion, we generated mice with postnatal, neuron-specific presenilin-1 (PS1) deficiency, denoted PS1(n−/−), that were viable and fertile, with normal brain morphology. In adult PS1(n−/−) mice, levels of endogenous brain amyloid peptides were strongly decreased, concomitant with accumulation of amyloid precursor protein (APP) C-terminal fragments. In the cross of APP[V717I]xPS1 (n−/−) double transgenic mice, the neuronal absence of PS1 effectively prevented amyloid pathology, even in mice that were 18 months old. This contrasted sharply with APP[V717I] single transgenic mice that all develop amyloid pathology at the age of 10–12 months. In APP[V717I]xPS1 (n−/−) mice, long-term potentiation (LTP) was practically rescued at the end of the 2 hr observation period, again contrasting sharply with the strongly impaired LTP in APP[V717I] mice. The findings demonstrate the critical involvement of amyloid peptides in defective LTP in APP transgenic mice. Although these data open perspectives for therapy of AD by γ-secretase inhibition, the neuronal absence of PS1 failed to rescue the cognitive defect, assessed by the object recognition test, of the parent APP[V717I] transgenic mice. This points to potentially detrimental effects of accumulating APP C99 fragments and demands further study of the consequences of inhibition of γ-secretase activity. In addition, our data highlight the complex functional relation of APP and PS1 to cognition and neuronal plasticity in adult and aging brain.

Expression of Human Apolipoprotein E4 in Neurons Causes Hyperphosphorylation of Protein Tau in the Brains of Transgenic Mice

Epidemiological studies have established that the epsilon 4 allele of the ApoE gene (ApoE4) constitutes an important risk factor for Alzheimers disease and might influence the outcome of central nervous system injury. The mechanism by which ApoE4 contributes to the development of neurodegeneration remains unknown. To test one hypothesis or mode of action of ApoE, we generated transgenic mice that overexpressed human ApoE4 in different cell types in the brain, using four distinct gene promoter constructs. Many transgenic mice expressing ApoE4 in neurons developed motor problems accompanied by muscle wasting, loss of body weight, and premature death. Overexpression of human ApoE4 in neurons resulted in hyperphosphorylation of the microtubule-associated protein tau. In three independent transgenic lines from two different promoter constructs, increased phosphorylation of protein tau was correlated with ApoE4 expression levels. Hyperphosphorylation of protein tau increased with age. In the hippocampus, astrogliosis and ubiquitin-positive inclusions were demonstrated. These findings demonstrate that expression of ApoE in neurons results in hyperphosphorylation of protein tau and suggests a role for ApoE in neuronal cytoskeletal stability and metabolism.

Prominent Cerebral Amyloid Angiopathy in Transgenic Mice Overexpressing the London Mutant of Human APP in Neurons

Deposition of amyloid beta-peptide (Abeta) in cerebral vessel walls (cerebral amyloid angiopathy, CAA) is very frequent in Alzheimers disease and occurs also as a sporadic disorder. Here, we describe significant CAA in addition to amyloid plaques, in aging APP/Ld transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP) exclusively in neurons. The number of amyloid-bearing vessels increased with age, from approximately 10 to >50 per coronal brain section in APP/Ld transgenic mice, aged 13 to 24 months. Vascular amyloid was preferentially deposited in arterioles and ranged from small focal to large circumferential depositions. Ultrastructural analysis allowed us to identify specific features contributing to weakening of the vessel wall and aneurysm formation, ie, disruption of the external elastic lamina, thinning of the internal elastic lamina, interruption of the smooth muscle layer, and loss of smooth muscle cells. Biochemically, the much lower Abeta42:Abeta40 ratio evident in vascular relative to plaque amyloid, demonstrated that in blood vessel walls Abeta40 was the more abundant amyloid peptide. The exclusive neuronal origin of transgenic APP, the high levels of Abeta in cerebrospinal fluid compared to plasma, and the specific neuroanatomical localization of vascular amyloid strongly suggest specific drainage pathways, rather than local production or blood uptake of Abeta as the primary mechanism underlying CAA. The demonstration in APP/Ld mice of rare vascular amyloid deposits that immunostained only for Abeta42, suggests that, similar to senile plaque formation, Abeta42 may be the first amyloid to be deposited in the vessel walls and that it entraps the more soluble Abeta40. Its ability to diffuse for larger distances along perivascular drainage pathways would also explain the abundance of Abeta40 in vascular amyloid. Consistent with this hypothesis, incorporation of mutant presenilin-1 in APP/Ld mice, which resulted in selectively higher levels of Abeta42, caused an increase in CAA and senile plaques. This mouse model will be useful in further elucidating the pathogenesis of CAA and Alzheimers disease, and will allow testing of diagnostic and therapeutic strategies.

Peroxisome-proliferator-activated receptor gamma induces a clearance mechanism for the amyloid-beta peptide.

We investigated whether peroxisome proliferator-activated receptor γ (PPARγ) could be involved in the modulation of the amyloid cascade causing Alzheimers disease. Inducing expression or activating PPARγ using synthetic agonists of the thiazolinedione family results in a dramatic decrease in the levels of the amyloid-β (Aβ) peptide in the conditioned medium of neuronal and non-neuronal cells. PPARγ does not affect expression or activity of any of the secretases involved in the generation of the Aβ peptide but induces a fast, cell-bound clearing mechanism responsible for the removal of the Aβ peptide from the medium. Although PPARγ expression is generally low in the CNS, induction of PPARγ expression during inflammation could be beneficial for inducing Aβ clearance. We confirm that the Aβ clearance mechanism can indeed be induced by PPARγ activation in primary murine-mixed glia and cortical neuronal cultures. Our results suggest that PPARγ-controlled mechanisms should be explored further as potential drug targets for Alzheimers disease treatment.

Presenilin 1 mediates the turnover of telencephalin in hippocampal neurons via an autophagic degradative pathway

Presenilin 1 (PS1) interacts with telencephalin (TLN) and the amyloid precursor protein via their transmembrane domain (Annaert, W.G., C. Esselens, V. Baert, C. Boeve, G. Snellings, P. Cupers, K. Craessaerts, and B. De Strooper. 2001. Neuron. 32:579–589). Here, we demonstrate that TLN is not a substrate for γ-secretase cleavage, but displays a prolonged half-life in PS1−/− hippocampal neurons. TLN accumulates in intracellular structures bearing characteristics of autophagic vacuoles including the presence of Apg12p and LC3. Importantly, the TLN accumulations are suppressed by adenoviral expression of wild-type, FAD-linked and D257A mutant PS1, indicating that this phenotype is independent from γ-secretase activity. Cathepsin D deficiency also results in the localization of TLN to autophagic vacuoles. TLN mediates the uptake of microbeads concomitant with actin and PIP2 recruitment, indicating a phagocytic origin of TLN accumulations. Absence of endosomal/lysosomal proteins suggests that the TLN-positive vacuoles fail to fuse with endosomes/lysosomes, preventing their acidification and further degradation. Collectively, PS1 deficiency affects in a γ-secretase–independent fashion the turnover of TLN through autophagic vacuoles, most likely by an impaired capability to fuse with lysosomes.

The Orphan G Protein–Coupled Receptor 3 Modulates Amyloid-Beta Peptide Generation in Neurons

Deposition of the amyloid-β peptide is a pathological hallmark of Alzheimers disease. A high-throughput functional genomics screen identified G protein–coupled receptor 3 (GPR3), a constitutively active orphan G protein–coupled receptor, as a modulator of amyloid-β production. Overexpression of GPR3 stimulated amyloid-β production, whereas genetic ablation of GPR3 prevented accumulation of the amyloid-β peptide in vitro and in an Alzheimers disease mouse model. GPR3 expression led to increased formation and cell-surface localization of the mature γ-secretase complex in the absence of an effect on Notch processing. GPR3 is highly expressed in areas of the normal human brain implicated in Alzheimers disease and is elevated in the sporadic Alzheimers disease brain. Thus, GPR3 represents a potential therapeutic target for the treatment of Alzheimers disease.

Insect neuropeptide F (NPF)-related peptides: Isolation from colorado potato beetle (Leptinotarsa decemlineata) brain

Two novel neuropeptides with neuropeptide F (NPF)-like immunoreactivity have been isolated from brain extracts of the Colorado potato beetle. Purification was achieved primarily by use of reverse phase chromatography including initial C-18 Sep-Pak cartridges and 4 subsequent analytical HPLC columns. Combined data from automated Edman degradation, immunochemical analysis, u.v. absorbance and mass spectrometry led to the elucidation of their full primary structures. The deduced sequences are: Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-NH2 (ARGPQLRLRFamide) and Ala-Pro-Ser-Leu-Arg-Leu-Arg-Phe-NH2 (APSLRLRFamide). On the basis of their primary structure both peptides can be appended to the invertebrate group of neuropeptide Y (NPY)-like peptides, generally referred to as NPFs. We suggest these peptides to be designated Led-NPF-1 and Led-NPF-2.

Neonatal neuronal overexpression of glycogen synthase kinase-3β reduces brain size in transgenic mice

Glycogen synthase kinase-3beta (GSK-3beta) is important in neurogenesis. Here we demonstrate that the kinase influenced post-natal maturation and differentiation of neurons in vivo in transgenic mice that overexpress a constitutively active GSK-3beta[S9A]. Magnetic resonance imaging revealed a reduced volume of the entire brain, concordant with a nearly 20% reduction in wet brain weight. The reduced volume was most prominent for the cerebral cortex, without however, disturbing the normal cortical layering. The resulting compacted architecture was further demonstrated by an increased neuronal density, by reduced size of neuronal cell bodies and of the somatodendritic compartment of pyramidal neurons in the cortex. No evidence for apoptosis was obtained. The marked overall reduction in the level of the microtubule-associated protein 2 in brain and in spinal cord, did not affect the ultrastructure of the microtubular cytoskeleton in the proximal apical dendrites. The overall reduction in size of the entire CNS induced by constitutive active GSK-3beta caused only very subtle changes in the psychomotoric ability of adult and ageing GSK-3beta transgenic mice.