S.16.01 Designing stem cells as therapeutic agents inneurological disease

S142

£16, The prosper*for stem cell therapy in neuropsyehopharmaeology

IS.15.05] PPI, habituation and sensitization of startle reflexes in schizophrenia U, Meincke 1, K. Heekeren 2, E. Gouzoulis-Mayfrank 2 . lI'2li~ikum NiederberE, PsycZ~iatry aud Psychotherapy, Ve.lbert, Germarry; 2 University Cologne, ?sychiatry and Psychotherapy, Cologne, Germany Abnormalities of attention and information processing are regarded as important features of schizophrenia. Theoretically, they may reflect deficiencies in central mechanisms of inhibition and selection. These deficiencies are believed to lead to cognitive fragmentation in patients with schizophrenia. In different paradigms (prepulse inhibition, habituation, sensitization), the acoustic startle reflex provides a tool to assess preattentive, i.e., automatic attention deficits in schizophrenia: Prepulse inhibition (PPT) of the startle reflex is an operational measure of sensorimotor gating that serves to automatically filter out irrelevant or interfering stimuli. The startle reflex consists of flexion and extension responses to sudden intense sensory stimuli. Usually, the blink response is measured electromyographically as the most stable and reliable component of the overall startle reaction. PPI is the reduction of the startle response when a relatively weak sensory stimulus is applied about 30-200 ms prior to the strong startling stimulus. In numerous studies schizophrenia patients were found to exhibit deficits in PPI. However, the trait or state nature of these impairments is unclear: Deficits were reported in drug-naive schizophrenia patients, in acutely decompensated inpatients and in stable out:patients with schizophrenia, in asymptomatic first-degree relatives of schizophrenic patients, in patients with schizotypal personali~ disorder, and in presumably psychosis-prone individuals, but, on the other hand, PPI impairment appeared to be associated with current positive symptoms, particularly formal thought disorder and distractibility, these latter findings suggesting a state dependence of PPI deficits in schizophrenia. In addition, PPI may be influenced by antipsychotic medication. In a follow up stu@ of 36 schizophrenia patients with an acute psychotic episode we found significant PPI deficits only in the acute state, but not in the remitted state. In addition, PPI deficits were associated with more severe formal thought disorder and bizarre behavior. Taken into account results of other studies in the field we propose that PPI impairment represent a mediating vulnerability marker of schizophrenia: it predisposes individuals to develop schizophrenic psychosis, but also covaries with the presence of acute positNe symptoms. In contrast to PPI, habituation deficits of the acoustic startle, i.e. impaired reflex decrement to repeated application of identical stimuli, have been demonstrated less consistently in schizophrenia patients. Reduced habituation in schizophrenia reflects impaired gating of repeatedly presented trivial stimuli that may result in cognitive fragmentation by sensory overload. As in many other studies, we were not able to detect significant deficits in a longitudinal study of acute schizophrenic patients using a block-to-block-analysis that allows also for an assessment of PPI in the same startle session. Nevertheless, applying a trialby-trial analysis we demonstrated an increased sensitization effect that had obscured habituation deficits in our sample of schizophrenia patients. According to the dual-process theory of Groves and Thompson (1970) sensitization is defined as an opposing, independent and superimposed incremental process that results in an initial increment of reflex magnitudes during a train of intense aversive stimuli. This abnormal increased sensitization effect in schizophrenia may reflect dysfunctional arousal modulation and may have confounded correct assessment of habituation processes

in studies using block-to-block analyses. In addition, exaggerated sensitization and reduced habituation in schizophrenia patients were not associated with psychopathological symptoms in our study and appeared therefore as trait markers of schizophrenia. These parameters reflect dysfunctional basic information processing that predisposes subjects to schizophrenic symptoms.

References [1 ] BraffDL, Geye" MA, SWerdlowNR (2001): Human studies ofprepulse inhibition of startle: normal subjects, patient groups, and pharmacological studies. Psydaopharmaool (Bed) 156: 234-258. [2] Geyer MA, BraffDL (1982): Habituation of the blink reflex in norrnals and s&izophrenlo patients. Psyohophysiol 19:1-6. [3] Groves PM, Thompson RF (1970): Habituation: A dual process theory. Psy&ol Reu 77: 41%450. [4] Luderc~igK, Geyer MA, Etzensberger M, Vollenv~ider FX (2002) Stability of the acoustic startle refl~, prepulse inhibition, and habituation in s&izophr~ia. S&izophr Res 55: 12%137. [5] Melnolce U, Light GU, Geyer MA, Braff DL, Gouzoulis-Mayfrank E, Sensitization and habituation of the acoustic startle refl~ in patients with s&tizophrenia. Psy& Res (in press).

S.16. The prospect for stem cell therapy in neuropsychopharmacology ~

Designing stem cells as therapeutic agents in neurological disease

O, Isacson. Harvard Mediccg Sol,coL Neurology and Neuroscience, Belmont, MA, U~S,A,

Purpose of the study: Although progressive neurodegenerative diseases have very different and highly specific causes, the dysfunction or loss of a vulnerable group of neurons is common to all these disorders and rray allow the development of similar therapeutic approaches to the treatment of diseases such as amyotrophic lateral sclerosis, Parkinson's disease, and Huntington's disease. There are known molecular mechanisms and processes in stem and progenitor cells that may be of use in the future design and selection of cell-based replacement therapies for neurological diseases. Our aim is to provide an embryonic stem cell derived dopamine neuron that will be a genetically stable, homogeneous cell population to be used as a specific, functional and safe cell therapy for Parkinson's disease (PD). Methods: We have developed methodology to obtain transplantable dopamine neurons of the cell type that specifically degenerates in the midbrain of PD. t)y successive differentiation of embryonic stem cells (mouse, primate and human), we can obtain neuronal cell types in vivo or in vitro that can integrate Nnctionally in brain circuitry. In defined media and by step-wise culturing methods, neural marker defined cell populations (e.g. 17rosine hydroxylase, Nurrl and PitX3 positive dopamine neurons) can be isolated and prepared for cell transplantation to animal models of neurodegenerative diseases. For e~ample, dopamine neurons can be obtained and transplanted with functional recovery seen in PD animal models. Cell sorting methodology, combined with genetic engineering of fluorescent markers for the desired cell type or cell surface marker provide techniques to select cells for effective cell based therapies. Results" We demonstrate the molecular signals required for successful differentiation of embryonic stem cells into specific and functional midbrain dopamine neurons, which in turn can be transplanted in Parkinson's disease models. We have also evaluated alternative methods of restoring dopamine cells to the circuitry by

S.7 7, AntiFsychotics in anziety and affeetive disorders stimulation of the brain's endogenous stem cells by chemical and trophic factors. The development of stem cell based repair from dopamine neurons has been evaluated from the point of view of safety, such as preventing the formation of unwanted cell types that may interfere with neural graft function. For this purpose, we have tested various gent knockout and knockin embryonic stem cells in cell replacement paradigms. We find that fluorescenceactivated cell sorting or equivalent cell selection procedures are necessary steps for an adequate and appropriate surgical dopamine neuronal cell replacement therapy method. C o n c i s i o n : There are many lessons learned from the conceptual and clinical problems that have been encountered in the development of cell-based treatments, and specific criteria for the effective use of cells in the future treatment of nenrodegenerative diseases. A stem cell derived dopamine neuron will provide a more homogeneous population of cells, and a more controlled cell therapy for PD that allows epeeification of cell types and cell numbers transferred to the patients. Unwanted cell types in current fetal transplants, including vascular immunogenic cells, can be eliminated by a stem cell derived cell therapy and cell sorting. Future successful clinical trials using cell therapy for PD will require continued rational development, cell formulation and sophisticated use of stem cell technology. References

[1] Isaoson O,, Bjorklund L,M,, Sohumaches J,M (2003) Towards full restoration of synaptio and terminal function of the dopaminergie system in Parkinsen's disease from regeneration and neuronal r~lacemeut by stem cells. Ann. Nenrol. 537 $135-4g. [2] Isacaon O., Bjorldund L., Pernaute R.S. (2001) Parlcinson's disease: interpretations of transplantation study are en'eneens. Nat. Nenrosoi. 4, 553, [3] Bjorldund L., Pernaute R.S., Chung S., Andersson T., Ohm I.Y.C.~ MeNaught K.S.2, Brownell A.-L., Jenkins B.G., Wahlestedt C., Kim K.-S., Isaeson O. (2002) Embryonic stem cells develop into functional dopaminergie neurons after transplantation in a Parkinson rat model. Proe. Natl. Acad. Sot. 99, 2344-2349. [4] Isacson, O. (2003) The production and use of calls as therapeutic agents in neurodegenerative diseases. Lancet Neurology~ 2, 417-424. [5] Chung, S., Senntag K-C., Andersson, 12, Bjorklund, L.M., Park, J.J., Kim, D-W., Kang, LIJ., Isacson, O., Kim, K.-S. (2002) Genetic engineering of mouse embryonic stern ceils by Nurrl enhances differentiation and maturation into dopaminergio neurons. Eur. J. Nenrosd. 16, 1829-1838.

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Neural stem cell and brain repair

A, Bjorklund. Wallenberg Neuroscience Center, DeFt of .P/Wsiological Sciences, Lurid University, Lurid Sweden 2-n v#ro-expanded multipotent neural stem- or progenitor ceils possess the capacity to differentiate into both neuronal and glial phenotypes both in vitro and after transplantation to the brain, in vice. We have previously shown that grafted neural progenitors can undergo extensive migration and integration into the host brain, and differentiate into cells that express both morphological and chemical features of mature neurons. Cellular neuronal markers, such as N o u n or neurofilament proteins, are commonly used to identify ceils that have differentiated toward a neuronal fate. It remains unclear, however, to what extent grafted neural progenitors can undergo full neuronal differentiation, develop physiological characteristics of functional neurons, and become functionally integrated into the neural circuitry of the host. Tn the present study we have used single-cell recording to characterize the electrophysiological properties of intracerebrally transplanted cells derived from a conditionally immortalized neural progenitor cell line,

g143

RN331;, and traced their axonal projections using FioroGold (FG) as a retrograde neuronal tracer. The RN33B cell line, which was generated from embryonic rat bralnstem by retroviral transduction of the temperature-sensitive SV40 laNe-T-antigen (Whittemore and White, 1993), has a remarkable neurogenic capacity and can differentiate into neuron-like cells with the morphology of both pyramidal cells and interneurons after transplantation into cortex or hippoeampus, and into medium-sized densely-opt W neurons after transplantation to the striatum. To label the grafted cells, we used a lentiviral vector to stably transduced the RN33B cells with Green Fluorescent Protein (GPF), which makes it possible to identify morphologically differentiated graft, cells by their native fluorescence for electrophysiological recordings. The GFI ) marker, moreover, allows visualization of the grafted cells in their entirety, including the fine details of their axons and dendrites in vice. Part of the GFP-expressing grafted cells differentiated into cells with morphological features of cortical and hippocampal pyramidal neurons, and established axonal connections with both thalamus and contalateral hippocampus. Recording from identified GFP-positive cells using whole-cell pat@ recording in cortical slices showed physiological properties similar to host pyramidal neurons, and evidence of Nnctional excitatory and inhibitory synaptio inputs from the host, indicating that grafted progenitors can differentiate into mature projection neurons. These data strongly suggest that neuronal differentiation from grafted neural progenitors is controlled by the presence of, as yet unidentified, local cues at the site of cell integration, and that direct cell-to-cell interactions, rather than diffusible molecules, are involved. The ability ofgraft.ed stern/progenitor cell lines to produce Nnctionally integrated neurons in areas outside the classic neurogenic brain regions, moreover, suggests that such cells may be useN1 for neuronal replacement and brain repair.

References [1] Bjhrklund, A and Lindvall, O. (2000) Cell Replacement Therapies for CNS Disorders, Nature Nenrosoi. 3:53%544. [2] EngNnd U., Fri&er-Gates R. A., Lundberg C., BjOrkNnd A., and Wictorln K. (2002) Transplantatlon of human n~ral progenitor calls into the neonatal rat brain: e~tensive migration and differentiation with long-distance axonal projeetiens. E×p Neurol. 173, 1-21. [3] Lundberg C, Englund U, Trono D, Bjhrklund a and Wiotorin K, (2002) Differentiation of the RN33B cell line intor forebrain projection neurons after transplantation into theneonatal rat brain, E×p Neurology, 175, 370-387. [4] Enginnd U, gjOrldund A, Wictorin K, Lindvall O, Kokaia M (2002) Grafted neural stem ceils develop into functional pyramidal neurons and integrate into host cortical circuitry. Proo Natl Aoad Set U S A. 99, 17089-17094, [5] Eriksson C, Bjorklund A, Wiotorin K, (2003) Neuronal differentiation following transplantation of expanded mouse neurosphere oultares derived from different embryonic forebrain regions. Exp Nenrol. 184(2):615-35.

S.17. Antipsychotics in anxiety and affective disorders ~

Atypical antipsychotics in social anxiety (SAD) and posttTaumatic stress disorder (PTSD)

J. Davidson. Duke University Medical Center, DeFartme~ of Psychiatry, Durham, NC, US, A, Rates of response to first line treatment of SAD and PTSD range between 40-70%, with remission rates varying from 15-30%. It is thus obvious that, in order to achieve optimal response, many