Max Holzer

Abundant Tau Filaments and Nonapoptotic Neurodegeneration in Transgenic Mice Expressing Human P301S Tau Protein

The identification of mutations in the Tau gene in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has made it possible to express human tau protein with pathogenic mutations in transgenic animals. Here we report on the production and characterization of a line of mice transgenic for the 383 aa isoform of human tau with the P301S mutation. At 5–6 months of age, homozygous animals from this line developed a neurological phenotype dominated by a severe paraparesis. According to light microscopy, many nerve cells in brain and spinal cord were strongly immunoreactive for hyperphosphorylated tau. According to electron microscopy, abundant filaments made of hyperphosphorylated tau protein were present. The majority of filaments resembled the half-twisted ribbons described previously in cases of FTDP-17, with a minority of filaments resembling the paired helical filaments of Alzheimers disease. Sarkosyl-insoluble tau from brains and spinal cords of transgenic mice ran as a hyperphosphorylated 64 kDa band, the same apparent molecular mass as that of the 383 aa tau isoform in the human tauopathies. Perchloric acid-soluble tau was also phosphorylated at many sites, with the notable exception of serine 214. In the spinal cord, neurodegeneration was present, as indicated by a 49% reduction in the number of motor neurons. No evidence for apoptosis was obtained, despite the extensive colocalization of hyperphosphorylated tau protein with activated MAP kinase family members. The latter may be involved in the hyperphosphorylation of tau.

Glutaminyl cyclase inhibition attenuates pyroglutamate Aβ and Alzheimer's disease–like pathology

Because of their abundance, resistance to proteolysis, rapid aggregation and neurotoxicity, N-terminally truncated and, in particular, pyroglutamate (pE)-modified Aβ peptides have been suggested as being important in the initiation of pathological cascades resulting in the development of Alzheimers disease. We found that the N-terminal pE-formation is catalyzed by glutaminyl cyclase in vivo. Glutaminyl cyclase expression was upregulated in the cortices of individuals with Alzheimers disease and correlated with the appearance of pE-modified Aβ. Oral application of a glutaminyl cyclase inhibitor resulted in reduced Aβ3(pE)–42 burden in two different transgenic mouse models of Alzheimers disease and in a new Drosophila model. Treatment of mice was accompanied by reductions in Aβx–40/42, diminished plaque formation and gliosis and improved performance in context memory and spatial learning tests. These observations are consistent with the hypothesis that Aβ3(pE)–42 acts as a seed for Aβ aggregation by self-aggregation and co-aggregation with Aβ1–40/42. Therefore, Aβ3(pE)–40/42 peptides seem to represent Aβ forms with exceptional potency for disturbing neuronal function. The reduction of brain pE-Aβ by inhibition of glutaminyl cyclase offers a new therapeutic option for the treatment of Alzheimers disease and provides implications for other amyloidoses, such as familial Danish dementia.

Expression of the cyclin-dependent kinase inhibitor p16 in Alzheimer's disease

RECENT evidence indicates that an impairment of neuronal adaptive and reparative processes might be a key feature of the pathomechanism of Alzheimers disease. Aberrations in the reparative neuronal response are likely to result from alterations of intracellular signal transduction cascades. As a consequence of abnormal intracellular signalling, the activation of those molecular events might be triggered in neurones which, in dividing cells, would lead to cellular transformation. In the present study, changes in the expression of the cyclin-dependent kinase inhibitor p16, a regulator of the orderly progression through the cell cycle, were investigated by immunocytochemical methods in the temporal cortex of patients with Alzheimers disease. Both neurofibrillary tangles and neuritic components of plaques showed strong p16 immunoreactivity. These findings support the hypothesis that an aborted attempt of terminally differentiated neurones to re-enter the cell cycle might be a critical event in the pathology of Alzheimers disease.

Expression of endothelial and inducible NOS-isoforms is increased in Alzheimer’s disease, in APP23 transgenic mice and after experimental brain lesion in rat: evidence for an induction by amyloid pathology

The nitric oxide-synthesizing enzyme nitric oxide synthase (NOS) is present in the mammalian brain in three different isoforms, two constitutive enzymes (i.e., neuronal, nNOS, and endothelial eNOS) and one inducible enzyme (iNOS). All three isoforms are aberrantly expressed in Alzheimers disease giving rise to elevated levels of nitric oxide apparently involved in the pathogenesis of this disease by various different mechanisms including oxidative stress and activation of intracellular signalling mechanisms. It still is a matter of debate, however, whether the abnormal expression of NOS isoforms has some primary importance in the pathogenetic chain and might thus be a potential therapeutic target or only reflects a secondary effect that occurs at more advanced stages of the disease process. To tackle this question, we analysed the expression of both eNOS and iNOS in patients with sporadic AD, in transgenic mice expressing human amyloid precursor protein (APP) with the Swedish double mutation under control of the Thy1 promotor (APP23 mice), and after electrolytic cortical lesion in rat, an experimental paradigm associated with elevated expression of APP. In all three conditions, an astrocytosis was induced accompanied by a strong increase of both iNOS and eNOS. Both NOS isoforms were frequently though not always colocalized. Thus, based on the expression pattern of NOS isoforms three types of astrocytes, expressing only one of the two isoforms or both together could be distinguished. In both AD and transgenic mice eNOS-expressing astrocytes exceeded iNOS-expressing astrocytes in number. Astrocytes with elevated levels of iNOS or eNOS were constantly seen in direct association with Abeta-deposits in AD and transgenic mice and were found in the vicinity of the lesion site in the rat cortex. The results of the present study show that expression of both iNOS and eNOS is increased in activated astrocytes under experimental conditions associated with elevated expression of APP (electrolytic brain lesion) or Abeta-deposition (APP23 transgenic mice). Therefore, it is suggested that altered expression of these NOS isoforms being part of AD pathology is secondary to the amyloid pathology and might not be primarily involved in the pathogenetic chain though it might contribute to the maintenance, self-perpetuation and progression of the neurodegenerative process.

Paired helical filament-like phosphorylation of tau, deposition of β/A4-amyloid and memory impairment in rat induced by chronic inhibition of phosphatase 1 and 2A

Alzheimers disease is histopathologically characterized by neurofibrillary tangles, formed by the abnormally high phosphorylated tau protein, and senile plaques which largely consist of the beta/A4-amyloid peptide. Metabolism of the amyloid precursor protein and its processing into beta/A4-amyloid is regulated by protein phosphorylation. Thus, an imbalance between protein phosphorylation and dephosphorylation might be crucial for the development of the molecular hallmarks of Alzheimers disease. We report here that chronic infusion into rat brain ventricles of okadaic acid, a specific inhibitor of the serine/threonine protein phosphatases 1 and 2A, results in a severe memory impairment, accompanied by a paired helical filament-like phosphorylation of tau protein and the formation of beta/A4-amyloid containing plaque-like structures in gray and white matter areas.

Neuronal expression of cycline dependent kinase inhibitors of the INK4 family in Alzheimer's disease

Summary. Neurodegeneration and cell death in Alzheimers disease might be associated with aberrant proliferative mechanisms and activation of cell-cycle related events. We reported previously on the elevated expression of the cyclin dependent kinase inhibitor p16INK4a in Alzheimers disease closely associated with neurofibrillary degeneration. In the present study, we demonstrate that other members of the INK4-family of cyclin dependent kinase inhibitors such as p15INK4b, p18INK4c and p19INK4d that bind directly to cdk4/6 or to complexes of cdk4/6 with D-type cyclins are all elevated. In contrast, no indication of altered expression of the cyclin dependent kinase inhibitors p21Cip1 and p27Kip1 were observed. Inhibitors of the INK4-family were strongly expressed in tangle-bearing neurones and neuritic components of plaques. A much lower expression was also seen in astrocytes. These findings add further evidence to the suggestion that a dysfunction of cell cycle regulation is of critical importance in the pathomechanism of Alzheimers disease.

Alzheimer-related τ-pathology in the perforant path target zone and in the hippocampal stratum oriens and radiatum correlates with onset and degree of dementia

Abnormal phosphorylation of the tau-protein is regarded as a crucial step in the formation of neurofibrillary tangles in the neuronal cell body and neuropil threads in dendrites. We studied the effects of tau-pathology on the clinical expression of dementia in 106 autopsy cases in the entorhinal region, the hippocampal stratum oriens, the stratum radiatum, and the perforant path target zone. The first cytoskeletal lesions were located in the perikarya and dendrites of the pre-alpha cells of the transentorhinal and entorhinal region. Next, abnormally phosphorylated tau-protein (PHF-tau) was found in the neuropil of the CA1-subiculum region. Thereafter, the stratum radiatum and stratum oriens began to be involved in PHF-tau pathology in Braak stage II. In the Braak stages IV and V, the stratum radiatum was completely involved, the stratum oriens increasingly so. Beginning in Braak stage III, we noted cases having PHF-tau pathology in the perforant path target zone of the outer molecular layer of the dentate gyrus. The increase of this pathology with ever greater involvement on the part of the entorhinohippocampal circuit correlated significantly not only with the Braak stages and with the neurochemically determined hippocampal content of PHF-tau but also with the degree of dementia as defined by the clinical dementia rating (CDR) scale. The affection of the stratum oriens in combination with PHF-tau pathology in the stratum radiatum and in the outer molecular layer of the dentate gyrus was encountered almost exclusively in demented individuals (CDR 1-3). These results indicate that axonal PHF-tau pathology in hippocampal pathways presumably is critical for the clinical expression of dementia and may constitute an anatomical substrate of clinically verifiable memory dysfunction in Alzheimers disease.

Pseudophosphorylation of tau protein alters its ability for self-aggregation

Filamentous tau protein deposits are a pathological hallmark of a group of neurodegenerative disorders (tauopathies). Tau protein in these aggregates is highly phosphorylated at different phosphorylation sites. Although tau filaments can be formed by heparin‐induced aggregation of unphosphorylated recombinant tau, it is not known how tau phosphorylation modulates aggregation behaviour. Analysis of the effect of tau phosphorylation at defined single or multiple sites is hampered by the low specificity of protein kinases and the highly dynamic turnover of phosphorylation in vivo. To overcome this problem we employed site‐directed mutagenesis to convert serine and threonine to aspartic acid or glutamic acid, which introduce a negative charge and conformational change that mimic phosphorylation. We tested 14 different mutated tau proteins for their propensity for self‐aggregation and formation of tau filaments. Tau aggregation was monitored with thioflavin S fluorescence in the presence of different inducers such as heparin, Al3+, Fe2+ and Fe3+. We found that mutations in the N‐terminal portion up to amino acid 208 mainly suppress tau aggregation, whereas mutations in the C‐terminal region mainly lead to an enhanced aggregation. Mutations in the middle portion of tau showed a mixed picture of suppression and enhancement of aggregation. A single amino acid change Ser422Glu has aggregation‐favouring properties with all four inducers.

Aberrant expression of nNOS in pyramidal neurons in Alzheimer's disease is highly co-localized with p21ras and p16INK4a.

Aberrancies of growth and proliferation-regulating mechanisms might be critically involved in the processes of neurodegeneration in Alzheimers disease (AD). Expression of p21ras and further downstream signalling elements involved in regulation of proliferation and differentiation as, for example, MEK, ERK1/2, cyclins, cyclin-dependent kinases and their inhibitors such as those of the p16INK4a family, are elevated early during the course of neurodegeneration. Activation of p21ras can also directly be triggered by nitric oxide (NO), synthesized in the brain by various isoforms of nitric oxide synthase (NOS) that might be differentially involved into the pathomechanism of AD. To study the potential link of NO and critical regulators of cellular proliferation and differentiation in the process of neurofibrillary degeneration, we analyzed the expression pattern of NOS-isoforms, p21ras and p16INK4a compared to neurofibrillary degeneration in AD. Additionally to its expression in a subtype of cortical interneurons that contain the nNOS-isoform also in normal brain, nNOS was detected in pyramidal neurons containing neurofibrillary tangles or were even unaffected by neurofibrillary degeneration. Expression of nNOS in these neurons was highly co-localized with p21ras and p16INK4a. Because endogenous NO can activate p21ras in the same cell which in turn leads to cellular activation and stimulation of NOS expression [H.M. Lander, J.S. Ogiste, S.F.A. Pearce, R. Levi, A. Novogrodsky, Nitric oxide-stimulated guanine nucleotide exchange on p21 ras, J. Biol. Chem. 270 (1995) 7017-7020], the high level of co-expression of NOS and p21ras in neurons vulnerable to neurofibrillary degeneration early in the course of AD thus provides the basis for an autocrine feedback mechanism that might exacerbate the progression of neurodegeneration in a self-propagating manner.

Tau and tauopathies.

Most neurodegenerative diseases are characterized by intracellular aggregates of insoluble proteins. As for the majority of these disorders, aetiology and pathogenesis are only poorly understood; current nosological concepts are largely based on these molecular signatures of protein aggregates which also provide valuable tools for neuropathological differential diagnosis. The microtubule associated protein tau is one of these proteins that form intracellular fibrillary deposits in neurons and glial cells of a large variety of disorders today collectively referred to as tauopathies. While dysfunction of tau has unequivocally been shown to be able to cause neurodegeneration, the precise mechanisms of how tau is involved in neurodegenerative disorders is still poorly understood. After research has focused for several decades on the axonal function of tau and on the fibrillar tau aggregation, more recent cell biological studies have opened up new insights into the role of tau at the synapse and in the nucleus. According to currently emerging cell biological concepts, tau might play a role in the regulation of neuronal plasticity in a wide array of neuronal networks. In addition, it might be involved in regulating genome stability. The most intriguing question relevant both to physiological and pathophysiological function of tau is the biological meaning of the large heterogeneity of isoforms of tau which apparently is a rather promiscuous molecule. The present review is divided into two parts. First, we give an overview on the molecular biology and cell biology of tau and its physiological functions. The second part deals with the pathophysiology of tau and description of tauopathies which comprise more than 20 disorders including Alzheimers disease, progressive supranuclear palsy, cortico basal degeneration, Picks disease and others.