Scientific contribution to the field of Research / Eigener wissenschaftlicher Beitrag zum Forschungsgebiet

Our research projects during the last ten years were focused on 1) the deposition of the amyloid b-protein (Ab) in the human brain, both in normal aging and Alzheimer’s disease (AD), and 2) on the deposition and toxicity of Ab in an amyloid precursor protein (APP)-transgenic mouse model for AD.

The deposition of Ab in the human brain

Comparing AD patients and non-demented elderly individuals for the numbers of senile plaques and their distribution within the medial temporal lobe we were able to show that the number of brain regions exhibiting Ab-plaques is increased in AD cases compared with non-demented elderly (6, 11). This increase is also paralleled with a progressive expansion of neurofibrillary tangle pathology as represented by the Braak-stages (10). The number of senile plaques, on the other hand, increases up to a maximum in early to mid stage AD-cases and then decreases in end-stage AD as indicated by the Braak-stage (4, 6). For the expansion of Ab-deposition in the medial temporal lobe, we showed that the different areas of the medial temporal lobe develop Ab-deposits in a distinct, hierarchical sequence (11). Thereby, all types of senile plaques were found to have their specific place in this sequence (11). Extending these observations to the entire human brain in serial 100µm thick sections of 1mm distance we have shown that the deposition of Ab in the human brain starts in the neocortex and expands step-by-step into further brain regions (16). This sequence allows the distinction of 5 phases (16). Comparing brains from non-demented elderly autopsy cases with that from AD cases, AD cases exhibited exclusively end stages of Ab-deposition whereas plaque deposition in the non-demented elderly was mainly restricted to early phases of Ab-deposition (16). Additional changes in the brain by other disorders, e.g. argyrophilic grain disease were associated with less severe AD-related Ab-pathology in demented cases (19). The deposition of senile plaques in the different brain areas often goes along with that of vascular Ab-deposits, i.e. with cerebral amyloid angiopathy (CAA). Vascular Ab-deposition starts in cortical and leptomeningeal vessels of the neocortex, and then expands further into vessels of allocortical areas and finally into that of subcortical areas (17). The expansion of CAA, thereby, parallels that of senile plaques. Both, the expansion of senile plaques and that of CAA correlate with one another (17). In so doing, we demonstrated that Ab-deposition in the human brain generally follows a hierarchical sequence in which the different regions become involved in senile plaque pathology as well as in vascular amyloid deposition (52). These results argue in favor of the hypothesis that Ab-deposition in non-demented elderly individuals represents the first step in the pathogenesis of AD rather than a normal phenomenon in the aging brain (16, 50). However, animal experiments were necessary to address the question whether the phases of Ab-deposition represent the time course of b-amyloidosis or just different pathologies in different individuals (see 2.2)).

In addition to these results, we described a new type of senile plaques, the fleecy amyloid (9). This plaque type is an early plaque form in the entorhinal region and in the CA1/subiculum field that is replaced by diffuse plaques in later stages (9, 11). Fleecy amyloid consists exclusively of Ab-deposits that exhibit the staining pattern of N-terminal truncated Ab (9). Fleecy amyloid as well as other types of plaques exhibiting the staining pattern of N-terminal truncated Ab co-localized apolipoprotein E (apoE) suggesting the presence of Ab – apoE complexes in these types of senile plaques (18). These plaques, especially the fleecy amyloid, were associated with high numbers of Ab-containing astrocytes (12). Therefore, it was tempting to speculate that these atrocytes remove apoE-linked forms of Ab from brain (12, 18). A clearance of apoE-linked forms of Ab by astrocytes would also explain the disappearance of the fleecy amyloid in later stages. A further argument that apoE is involved in the clearance of Ab is provided by our finding of a strong association of the apolipoprotein E (APOE) genotype with the presence or absence of capillary Ab-deposition (15). Capillary Ab-deposition was mainly restricted to carriers of an APOE e4-allele. The close association of the APOE e4-allele with capillary CAA led to the distinction of two types of CAA: type 1 = capillary CAA associated with the APOE e4-allele; and type 2 = non-capillary CAA associated with the absence of an APOE e4 allele (15).  In so doing, the APOE e4-associated deposition of Ab along capillaries may alter the blood brain barrier function for the clearance of Ab and lead to an increased accumulation of Ab in the brain. To further investigate possible ApoE-linked clearance mechanisms for Ab relevant for AD animal experiments were required (see 2.2)).

The deposition and toxicity of Ab in an APP-transgenic mouse model for AD

In the mouse model of the APP23 mouse overexpressing human APP harboring the Swedish mutation driven by the Thy-1 promoter, we found a similar sequence in which the different areas of the brain were involved in b-amyloidosis as in the human brain representing the time course of Ab-deposition in this mouse model (52). The progression of Ab-deposition was accompanied with progressive neuronal alterations which were detected with the DiI-tracing method (23).

In detail, by analyzing commissural neurons of the frontocentral cortex we were able to show first a degeneration of a distinct type of commissural neurons which was characterized by a highly ramified and prominent dendritic tree. Other types of commissural neurons were not altered at this point in time. 10 months later, a second type of commissural neurons showed degeneration while the third type did not show any alterations (23). In so doing, we were the first to show that Ab is capable of inducing selective neuronal degeneration of vulnerable neurons. The complexity of the dendritic arbor, thereby, appeared to explain selective vulnerability of neurons against Ab (23).

Characterizing the role of apoE in Ab-clearance of APP23 mice producing large amounts of Ab in the brain we found that Ab can be detected easily in the perivascular space of APP23 mice (22). Here, Ab did not show amyloid properties but co-localized apoE suggesting that apoE is involved in the perivascular drainage of Ab. In a mouse model expressing human apoE driven by a GFAP-promoter leading to an expression of human apoE only in astrocytes (22) we confirmed that astroglial-derived apoE is indeed subject of perivasular drainage regardless of its association with Ab (22). In other words, apoE plays an important role in extracellular fluid drainage along perivascular channels. Taken together, our results suggest that non-fibrillar forms of Ab are drained along perivascular channels and that apoE is presumably involved in this clearance mechanism. Overloading such a clearance mechanism in APP-transgenic mice appears to result in insufficient Ab-clearance, increased Ab-levels in the brain and the perivascular drainage channels, and finally in Ab-deposition. In so doing, our results strengthen the hypothesis that an alteration of perivascular drainage supports Ab-deposition and the development of AD.

Future Research Projects / Geplante Forschungsprojekte

In my future research projects I will continue studying Ab-deposition in the human tissue as well as in animal models for AD. The following projects are planned: 1) the establishment of a classification for Ab-deposition in neuropathological practice in collaboration with the BrainNet Europe II consortium, 2) the identification of Ab-forms associated with degenerating neurons, 3) the characterization of treatment effects of Ab-immunization on the neurodegenerative changes identified in commissural neurons of the frontocentral cortex, and 4) the clarification of the role of apoE in Ab-deposition and clearance.

The establishment of a classification for Ab-deposition in neuropathological practice in collaboration with the BrainNet Europe II consortium

To establish a classification for Ab-deposition that can be used in neuropathological practice especially for Brain-Banking purposes I am involved in the activities of the BrainNet Europe II consortium (supported by the EU) (45). The aims of my collaboration with the BrainNet Europe II are to establish a) guidelines for a reliable staining procedure for Ab-plaques and CAA that can be used in every laboratory without major variations, b) guidelines for neuropathologists enabling them to classify senile plaque lesions and cerebrovascular Ab-lesions without major inter-rater and intra-rater variability, and c) a classification that allows a ranked classification of the severity of Ab-lesions in the brain. The conceptual organization and the criteria for determining Ab-pathology to be tested are developed by a group of five “BrainNet Europe II” neuropathologists with expertise in the classification of Ab-changes (Irina Alafuzoff (Kuopio, Finland), Thomas Arzberger (Munich, Germany), Nenad Bogdanovic (Huddinge, Sweden), Hans Kretzschmar (Munich, Germany) and Dietmar R. Thal (Bonn, Germany)) chaired by Irina Alafuzoff. With these activities we plan to test whether the phases of Ab-deposition for senile plaques (16) as well as that for CAA (17) represent reliable ranking scales for the assessment of the severity of Ab-deposition and CAA in the human brain in inter-rater studies. Since I contributed significantly in the development of both staging systems, my major input in this program is the preparation of foolproof guidelines for the use of these classifications and to provide the determinations of the Ab-lesions that have to be diagnosed by the BrainNet Europe II neuropathologists in inter-rater trials. As soon as such a staging system is proven to be acceptable for the BrainNet Europe II-members I want to correlate the results of such an acceptable staging system for Ab with the clinical symptoms of the patients and Ab-imaging data before death.

Since today Ab-pathology is not covered by the recommended classification criteria for AD, the major goal of this project is to introduce a reliable and valid classification for Ab-pathology for brain-banking and research purposes.

The identification of Ab-forms associated with degenerating neurons (collaboration with M. Staufenbiel, Novartis, Basel, Switzerland)

One of the most important results of my recent research is the identification of selective vulnerability against Ab. To identify the forms of Ab leading to neurodegeneration we plan to stain vulnerable and non-vulnerable neurons with antibodies against Ab₄₀, Ab₄₂, Ab_1-17, oligomeric Ab, and fibrillar Ab at the electron microscopic level. Since in 5 months old APP-transgenic mice only one type of commissural neurons degenerates whereas the other types of commissural neurons do not it is possible to study vulnerable and non-vulnerable neurons of the same animal at the electron microscopic level using the DiI-tracing method with subsequent photoconversion for cell identification, and to analyze them for their association with the different forms of Ab. These observations will show us which form of Ab is the toxic one specifically occurring in association with vulnerable but not with non-vulnerable neurons. Moreover, these experiments will identify the major neuronal target of the toxic Ab-forms: the dendrites, the soma, or the axon. Whether intra- or extracellular Ab is required for its toxic effects and which ultrastructural compartments are involved will also be clarified with these observations as well as with additional cell culture experiments.

Characterization of treatment effects of Ab-immunization on the neurodegenerative changes identified in commissural neurons of the frontocentral cortex of APP-transgenic mice (collaboration with M. Staufenbiel, Novartis, Basel, Switzerland)

The third question I am going to address is whether treatment strategies aimed at reducing the Ab-load in the brain, such as Ab-immunization, enable protection for neurodegeneration and/ or recovery of degenerated neurons, such as the heavily ramified type of commissural neurons in APP-transgenic mice (23). This research project, therefore, is aimed at clarifying 1) whether neuronal changes due to Ab-induced neurodegeneration can be prevented by immunotherapy prior to the onset of the changes, and 2) whether they are potentially subject for recovery. Both aims are tackled by studying APP-transgenic mice treated before and/or after the onset of neurodegeneration. Ab-lowering treatment strategies will be active and passive Ab-immunization. DiI-tracing of frontocentral commissural neurons will be used to identify neurodegeneration as previously described (23). Recovery of degenerated neurons from Ab-induced degeneration is given in the event that the number and the morphology of commissural neurons is equal in APP23 mice treated after the onset of neurodegeneration and in wild-type controls but different from that in untreated APP23 mice. In the event that recovery is not seen, animals treated before the onset of neurodegeneration will be studied for the presumable protective effects of Ab-lowering treatments.

With these observations, I will be able to show whether Ab-immunization is capable of preventing degeneration of distinct cell types and whether regeneration is possible after treatment. These results will impact the use of immunization strategies and its future clinical application in AD treatment concepts.

Clarification of the role of apoE in Ab-deposition and clearance (Collaboration with F. van Leuven, Katholieke Universiteit, Leuven, Belgium)

Our recent results indicate together with those of other authors that apoE is involved in the development of newly formed plaques (18) and in the clearance of Ab along perivascular channels (22). To further study the role of apoE in astrocytes and neurons for the deposition and clearance of Ab, I will compare double transgenic mice for APP and apoE expressing apoE driven by an astrocyte specific GFAP-promoter with those expressing apoE driven by a neuron-specific Thy-1-promoter. Using double label immunofluorescence with antibodies detecting specifically human apoE and laser scan microscopy we want to address the questions 1) whether apoE and Ab already co-localize within the cells producing apoE or whether it interacts with Ab only in the extracellular space, 2) whether astrocytes take up Ab already linked to apoE in vivo, 3) whether the uptake of apoE-Ab-complexes leads to a dissociation of the complexes within the cell enabling enzymatic degradation of Ab, and 4) which cells represent the driving force for the deposition of presumable apoE-Ab complexes in newly formed plaques: astrocytes or neurons. The use of the two mouse models producing human apoE driven by 1) an astroglial- and 2) a neuron specific promoter enables us to follow the uptake and deposition of human apoE in neurons in those animals expressing human apoE only in astrocytes and to follow the uptake and deposition of human apoE in astrocytes in mice expressing this protein only in neurons. Additional cell culture experiments to study the uptake of exogenous Ab in astroglial cell lines will be performed as well. With these experiments we can clarify the role of astrocytes and neurons for the trafficking of apoE and apoE-Ab complexes into drainage channels and for the cellular clearance of apoE-Ab complexes.

Since the APOE e4-allele is a major risk factor for AD, these observations will help to understand the pathological function of apoE in AD and may provide new options for therapeutical intervention.

Literature on Request.

Wichtigste Publikationen:

1. Thal, D.R.; Rüb, U.; Schultz, C.; Sassin, I.; Ghebremedhin, E.; Del Tredici, K.; Braak, E.; Braak, H. Sequence of Ab-protein deposition in the human medial temporal lobe. J. Neuropathol. Exp. Neurol., 59 (2000) 733-748

2. Thal, D.R.; Ghebremedhin, E.; Rüb, U.; Yamaguchi, H.; Del Tredici, K.; Braak, H. Two types of sporadic cerebral amyloid angiopathy. J. Neuropathol. Exp. Neurol. 61 (2002) 282-293

3. Thal, D.R.; Rüb, U.; Orantes, M.; Braak, H. Phases of Ab-deposition in the human brain and its relevance for the development of AD. Neurology 58 (2002) 1791-1800

4. Thal, D.R.; Ghebremedhin, E.; Orantes, M.; Wiestler, O.D. Vascular pathology in Alzheimer disease: Correlation of cerebral amyloid angiopathy and arteriosclerosis/lipohyalinosis with cognitive decline. J. Neuropathol. Exp. Neurol. 62 (2003) 1287-1301

5. Thal, D.R.; Del Tredici, K.; Braak, H. Neurodegeneration in normal brain aging and disease. Sci. Aging Knowledge Environ. (2004) PE: 26

6. Schröder, R.; Watts, G.D.J.; Mehta, S.G.; Evert, B.O.; Broich, P.; Fließbach, K.; Pauls, K.; Hans, V.H.; Kimonis, V.; Thal, D.R. Mutant valosin-containing protein causes a novel type of frontotemporal dementia. Ann. Neurol. 57 (2005) 457-461.

7. Thal, D.R.; Capetillo-Zarate, E.; Del Tredici, K.; Braak, H. The development of amyloid b protein deposits in the aged brain. Sci. Aging Knowledge Environ. (2006) RE: 1

8. Capetillo-Zarate, E.; Staufenbiel, M.; Abramowski, D.; Haass, C.; Escher, A.; Stadelmann, C.; Yamaguchi, H.; Wiestler, O.D.; Thal, D.R. Selective vulnerability of different types of commissural neurons for amyloid b-protein induced neurodegeneration in APP23 mice correlates with dendritic tree morphology. Brain 129 (2006) 2992-3005

9. Thal, D.R.; Larionov, S.; Abramowski, D.; Wiederhold, K.-H.; van Dooren, T.; Yamaguchi, H.; Haass, C.; van Leuven, F.; Staufenbiel, M.; Capetillo-Zarate, E. Occurrence and co-localization of amyloid b-protein and apolipoprotein E in perivascular drainage channels of wild-type and APP-transgenic mice. Neurobiol. Aging 28 (2007) 1221-1230.

10. Kölsch, H.; Larionov, S.; Dedeck, O.; Orantes, M.; Birkenmeier, G.; Griffin, W.S.T.; Thal, D.R. Association of the glutathione S-transferase omega-1 Ala140Asp polymorphism with cerebrovascular atherosclerosis and plaque-associated interleukin-1a expression. Stroke 38 (2007) 2847-2850