Katharina Schindowski

Neurotrophic factors in Alzheimer's disease: role of axonal transport.

Neurotrophic factors (NTF) are small, versatile proteins that maintain survival and function to specific neuronal populations. In general, the axonal transport of NTF is important as not all of them are synthesized at the site of its action. Nerve growth factor (NGF), for instance, is produced in the neocortex and the hippocampus and then retrogradely transported to the cholinergic neurons of the basal forebrain. Neurodegenerative dementias like Alzheimer’s disease (AD) are linked to deficits in axonal transport. Furthermore, they are also associated with imbalanced distribution and dysregulation of NTF. In particular, brain‐derived neurotrophic factor (BDNF) plays a crucial role in cognition, learning and memory formation by modulating synaptic plasticity and is, therefore, a critical molecule in dementia and neurodegenerative diseases. Here, we review the changes of NTF expression and distribution (NGF, BDNF, neurotrophin‐3, neurotrophin‐4/5 and fibroblast growth factor‐2) and their receptors [tropomyosin‐related kinase (Trk)A, TrkB, TrkC and p75NTR] in AD and AD models. In addition, we focus on the interaction with neuropathological hallmarks Tau/neurofibrillary tangle and amyloid‐β (Abeta)/amyloid plaque pathology and their influence on axonal transport processes in order to unify AD‐specific cholinergic degeneration and Tau and Abeta misfolding through NTF pathophysiology.

p25/Cdk5-mediated retinoblastoma phosphorylation is an early event in neuronal cell death

In large models of neuronal cell death, there is a tight correlation between Cdk5 deregulation and cell-cycle dysfunction. However, pathways that link Cdk5 to the cell cycle during neuronal death are still unclear. We have investigated the molecular events that precede p25/Cdk5-triggered neuronal death using a neuronal cell line that allows inducible p25 expression. In this system, no sign of apoptosis was seen before 24 hours of p25 induction. Thus, at that time, cell-cycle-regulatory proteins were analysed by immunoblotting and some of them showed a significant deregulation. Interestingly, after time-course experiments, the earliest feature correlated with p25 expression was the phosphorylation of the retinoblastoma protein (Rb). Indeed, this phosphorylation was observed 6 hours after p25 induction and was abolished in the presence of a Cdk5 inhibitor, roscovitine, which does not inhibit the usual Rb cyclin-D kinases Cdk4 and Cdk6. Furthermore, analyses of levels and subcellular localization of Cdk-related cyclins did not reveal any change following Cdk5 activation, arguing for a direct effect of Cdk5 activity on Rb protein. This latter result was clearly demonstrated by in vitro kinase assays showing that the p25-Cdk5 complex in our cell system phosphorylates Rb directly without the need for any intermediary kinase activity. Hence, Rb might be an appropriate candidate that connects Cdk5 to cell-cycle deregulation during neuronal cell death.

Neurogenesis and cell cycle-reactivated neuronal death during pathogenic tau aggregation

The aim of the present study was to investigate the relation between neurogenesis, cell cycle reactivation and neuronal death during tau pathology in a novel tau transgenic mouse line THY‐Tau22 with two frontotemporal dementia with parkinsonism linked to chromosome‐17 mutations in a human tau isoform. This mouse displays all Alzheimer disease features of neurodegeneration and a broad timely resolution of tau pathology with hyperphosphorylation of tau at younger age (up to 6 months) and abnormal tau phosphorylation and tau aggregation in aged mice (by 10 months). Here, we present a follow‐up of cell cycle markers with aging in control and transgenic mice from different ages. We show that there is an increased neurogenesis during tau hyperphosphorylation and cell cycle events during abnormal tau phosphorylation and tau aggregation preceding neuronal death and neurodegeneration. However, besides phosphorylation, other mechanisms including tau mutations and changes in tau expression and/or splicing may be also involved in these mechanisms of cell cycle reactivation. Altogether, these data suggest that cell cycle events in THY‐Tau22 are resulting from neurogenesis in young animals and cell death in older ones. It suggests that neuronal cell death in such models is much more complex than believed.

Early Tau pathology involving the septo-hippocampal pathway in a Tau transgenic model: relevance to Alzheimer's disease.

Alzheimers disease is a neurodegenerative disorder characterized by amyloid deposits and neurofibrillary tangles. Cholinergic dysfunction is also a main pathological feature of the disease. Nevertheless, the links between cholinergic dysfunction and neuropathological hallmarks of Alzheimers are still unknown. In the present study, we aimed to further investigate Tau aggregation in cholinergic systems, in a Tau transgenic mouse model. THY-Tau22 mice have recently been described as a novel model of Alzheimer-like Tau pathology without motor deficits. This strain presents an age-dependent development of Tau pathology leading to synaptic dysfunctions as well as learning and memory impairments. In the present work, we observed that Tau pathology differentially affects cerebral structures. Interestingly, early Tau pathology was observed in both hippocampus and basal forebrain. Moreover, some morphological as well as functional alterations of the septohippocampal pathway suggest a disconnection between these two key brain regions in Alzheimers disease. Finally, these data suggest that Tau pathology may participate in cholinergic degeneration.

S3-03-06

not available. S3-04-02 NGF GENE THERAPY Mark H. Tuszynski, Leon Thal, Mary Pay, Gang Tong, Hoi-Sang U, Roy Bakay, Steve Potkin, Gilbert Ho, Raymond Bartus, Zoe Arvanitakis, David Bennett, David Salmon, University of California San Diego and VA Medical Center, La Jolla, CA, USA; University of California, San Diego, La Jolla, CA, USA; Rush University Medical Center, Chicago, IL, USA; University of California, Irvine, Irvine, CA, USA; Ceregene, Inc., San Diego, CA, USA. Contact e-mail: mtuszynski@ucsd.edu Background: Cholinergic neuron loss is a cardinal feature of Alzheimer disease (AD). Nerve Growth Factor (NGF) stimulates cholinergic function, improves memory and prevents cholinergic degeneration in animal models of injury, amyloid overexpression and aging. Objective: We performed a Phase 1 trial of ex vivo NGF gene delivery in eight mild AD patients to assess safety and feasibility. Methods: Autologous fibroblasts genetically modified to express human NGF were injected into the basal forebrain to act as long-term sources of growth factor production. Adverse effect profile, cognitive function and PET scans were serially monitored. Results: Two sedated but non-anesthetized subjects moved as cells were intracerebrally injected, causing bleeds; all subsequent injections performed under anesthesia were without complication. After follow-up of up to 4 years post-treatment, no long-term adverse effects of NGF have occurred. Evaluation of the MMSE and ADAS-Cog suggested that the rate of cognitive decline post-treatment was reduced approximately 50%, over a mean period of two years. Serial PET scans demonstrated significant increases in cortical 18-fluorodeoxyglucose after treatment. Brain autopsy from one subject demonstrated robust growth responses to NGF. Conclusions: Additional clinical trials of NGF for AD are warranted. A Phase 1 trial of AAV-NGF gene delivery is in progress, and Phase 2 trials are planned. S3-04-03 BYPASSING THE BLOOD-BRAIN BARRIER WITH INTRANASAL DELIVERY TO TREAT ALZHEIMER’S DISEASE AND RELATED DISORDERS William H. Frey II, Alzheimer’s Research Center, Regions Hospital, St Paul, MN, USA. Contact e-mail: Alzheimr@umn.edu Background: Intranasal delivery provides a practical, noninvasive, method of bypassing the blood-brain barrier to deliver therapeutic agents to the brain and spinal cord. [See Dhanda (2005) Drug Delivery Technology 5(4):64-72 for a review.] Objective(s): Intranasal delivery allows drugs that do not cross the blood-brain barrier to be delivered to the central nervous system (CNS) within minutes. It also directly targets drugs that do cross the blood-brain barrier to the CNS, eliminating the need for systemic delivery and thereby reducing unwanted systemic side effects. Methods: Intranasal delivery does not require any modification of the therapeutic agent. A wide variety of therapeutics, including both small molecules and macromolecules are rapidly delivered intranasally to the brain and can target the olfactory and connected memory areas affected by Alzheimer’s disease (AD). This is possible because of the unique connection that the olfactory and trigeminal nerves provide between the brain and external environment [Thorne (2004) Neuroscience 127:481-496]. Conclusions: Using the intranasal delivery method, which I first introduced in 1989, researchers in Italy have reversed neurodegeneration and rescued memory in a transgenic mouse model of AD [Capsoni (2002) PNAS 99(19):1243212437 and De Rosa (2005) PNAS 102(10): 3811-3816]. We and others have demonstrated both treatment of and protection against stroke in animals with intranasal IGF-I [Liu (2004) Journal of Stroke and Cerebrovascular Diseases 13(1): 16-23], deferoxamine [Panter (2005) Society for Neuroscience Abstracts #669.5] and EPO [Yu (2005) Neurosci Lett 387:5]. Researchers in Israel have used intranasal delivery to target NAP and ADNF to the brain to treat anxiety and neurodegeneration [Alcalay (2004) Neuroscience Letters 361:128-131; Gozes (2000) JPET 293(3):10911098.] Intranasal NGF and EGF have been shown to stimulate neurogenesis in adult animals [Jin (2003) Ann Neurol 53:405-409]. Our collaborators [Reger (2006) Neurobiology of Aging: in press] have demonstrated that intranasal insulin acutely improves memory in patients with AD and those with mild cognitive impairment while researchers in Germany [Benedict (2004) Psychoneuroimmunol. 29:1326-1334] have shown that eight weeks of intranasal insulin treatment improves both memory and mood in normal adult humans. This new method of delivery can revolutionize the treatment of Alzheimer’s disease, stroke, and other brain disorders. S3-04-04 IMMUNOTHERAPY Roger Nitsch, University of Zurich, Zurich, Switzerland. Contact e-mail: nitsch@bli.unizh.ch Abstract not available.not available. S3-04-05 PHAGE THERAPY IN ALZHEIMER’S DISEASE Beka Solomon, Tel-Aviv Univ, Ramat Aviv, Israel. Contact e-mail: beka@post.tau.ac.il Filamentous phages (Ff) are well understood at both structural and genetic levels. Unlike lytic phages which are released by cell lysis after assembly in the host cell cytoplasm, Ff phages are extruded through their E. coli host cells without affecting them. They have a filamentous appearance, with a long (900 nm) and narrow (7 nm) Ff virion consisting of a single-stranded (ss) DNA genome packaged in a tube comprising 2700 copies of the major coat protein pVIII and closed at the ends by four or five copies of each of four species of minor coat proteins, including pIII. We recently found that the linear structure of filamentous phages confers permeability through the membranes and anti-aggregating properties against -amyloid (A ). Modifying the phage’s linear structure and rendering it spherical abolished its disaggregating as well as penetrating abilities. Here we describe modulating effect of filamentous phage on in vitro aggregation of amyloidpeptide (A P) as well as its neuroprotective activity against aggregated amyloidtoxicity. In vivo, phage’s disaggregating properties of -amyloid plaques were demonstrated by intracerebral injection into transgenic mice over-expressing the human amyloid precursor protein (hAPP). The high permeability of Ff to different kinds of membranes enables them to be delivered to the brain and to serve as delivery vectors of antibodies/ antigens. No toxic effects were observed in the challenged animal sections, including brain, liver, kidney, spleen and lung, following phage administration. Repeated intranasal administration of phage alone and/or phage anti-A antibodies in Alzheimer’s disease (AD) transgenic mice led to reduction of the -amyloid load associated with improvement of cognitive functions of treated animals. The cumulative effect of anti-A P antibodies and of the phage in dissolving amyloid plaques and oligomers increases the S50 Symposia S3-04: Therapeutic Strategies

P1-062: Alzheimer’s disease-like tau neuropathology leads to memory deficits and loss of functional synapses in a novel mutated tau transgenic mouse without any motor deficits

Tau transgenic mice are valuable models to investigate the role of tau protein in Alzheimers disease and other tauopathies. However, motor dysfunction and dystonic posture interfering with behavioral testing are the most common undesirable effects of tau transgenic mice. Therefore, we have generated a novel mouse model (THY-Tau22) that expresses human 4-repeat tau mutated at sites G272V and P301S under a Thy1.2-promotor, displaying tau pathology in the absence of any motor dysfunction. THY-Tau22 shows hyperphosphorylation of tau on several Alzheimers disease-relevant tau epitopes (AT8, AT100, AT180, AT270, 12E8, tau-pSer396, and AP422), neurofibrillary tangle-like inclusions (Gallyas and MC1-positive) with rare ghost tangles and PHF-like filaments, as well as mild astrogliosis. These mice also display deficits in hippocampal synaptic transmission and impaired behavior characterized by increased anxiety, delayed learning from 3 months, and reduced spatial memory at 10 months. There are no signs of motor deficits or changes in motor activity at any age investigated. This mouse model therefore displays the main features of tau pathology and several of the pathophysiological disturbances observed during neurofibrillary degeneration. This model will serve as an experimental tool in future studies to investigate mechanisms underlying cognitive deficits during pathogenic tau aggregation.