Wolfgang Streit, PhD - US grants

DESCRIPTION: (Adapted from Applicant's Abstract): This is a resubmitted application to determine the role, if any, of microglia in neuronal protection and assistance in regeneration following injury. It has three specific aims. The first aim is to measure the cytokines produced by microglia during the regeneration of the facial nerve neurons following axotomy and during rubrospinal nucleus neuronal degeneration following lateral funiculotomy. By RT-PCR synthesis of a number of cytokines and growth factors will be assayed and the time course of their synthesis defined. The second specific aim is to determine if CGRP synthesized by injured neurons activates microglial cells and causes their proliferation, cytokine synthesis or neurotrophin production. This will be done by both histochemical and RT-PCR techniques. In the third aim he will compare and hopefully identify genes differentially expressed by microglial cells in vitro and ex vivo. He will compare unstimulated, LPS activated and CGRP-stimulated microglial cells in vitro. With microglial cells freshly isolated from the facial nucleus following different stimuli, he will compare resting microglia, phagocytic microglia and reactive/activated microglial cells. He will subsequently clone and/or identify by partial sequences the genes differentially expressed.

DESCRIPTION (provided by applicant): The causes that underlie primary degeneration of motoneurons in ALS remain unknown. This proposal will investigate a novel hypothesis regarding ALS pathogenesis, namely, that dysfunctional microglia are, in part, responsible for motoneuron degeneration. The rationale for this hypothesis is two-fold: on one hand experimental studies in rodents suggest that microglial activation is essential for rescuing acutely injured motoneurons and for facilitating their regeneration, and on the other hand there is now evidence that microglial cells undergo structural deterioration in the aged and, particularly, in the Alzheimer's disease brain. The latter observations suggest that progressive structural deterioration of microglia over long periods of time results in microglial malfunction and dwindling glial support to neurons, which ultimately causes neurodegeneration. We now propose to investigate whether dystrophic microglia are present in motor neuron disease (MND). The specific aims are as follows: 1) To examine post-mortem human tissue specimens from individuals who died with ALS to determine if the incidence of dystrophic microglial cells is higher than in age-matched non-ALS individuals. 2) To determine if dystrophic microglial cells are present in the recently generated transgenic ALS rat carrying the SOD1/G93A mutation. 3) To determine the effect of minocycline treatment on axotomy-induced microglial activation in wildtype Sprague Dawley rats. This last set of studies is intended to represent a proof-of-principle experiment, which will test recent claims that minocycline delays the onset of MND by inhibiting microglial activation. If this claim is true, an attenuation of axotomy-induced microglial activation with minocycline should result in delayed or impaired motoneuron regeneration after axotomy. Collectively, these studies may provide a new lead into the pathogenesis of ALS, i.e. that dystrophy and dysfunction of microglia may be involved. In addition, the minocycline experiments will help to further assess the usefulness of this drug for possible treatment of ALS.

DESCRIPTION (provided by applicant): Neuroinflammation, which is evident as microglial activation in both the normally aged brain and in the Alzheimer's disease (AD) brain, is thought to be a contributing factor in neurodegeneration. The cause(s) of glial neuroinflammation are unknown, since it occurs in the absence of obvious neuronal injury. In this proposal, we will investigate the novel hypothesis that microglial senescence accounts for the observed glial neuroinflammation in the aging brain. We postulate that microglial cells are subject to an age-related decline in cellular structure and function that results from multiple, interconnected etiologies, including structural deterioration of cell cytoplasm, increased apoptosis, replicative senescence, and telomere shortening and/or concomitantly altered telomerase activity. Thus, "diseased" microglia could be responsible, in part, for age-related neurodegenerative changes due to a progressive loss of glial trophic support for neurons over time. The specific aims are designed to investigate multiple aspects of the presumed microglial senescence and dysfunction using a variety of approaches that include human post-mortem tissue, experimental studies in rats, as welt as studies in cell culture. They are as follows: Specific Aim 1: To analyze morphological abnormalities associated with microglial cells in the human brain using post-mortem tissue from both AD and non-demented, age-matched individuals. Specific Aim 2: To determine whether microglia, the only mature cell type in the CNS with significant mitotic potential, are subject to replicative senescence. These studies are to be performed in young and old rats using the facial nerve system as a reproducible model for stimulating microglial mitosis. Specific Aim 3: To determine whether microglial cells undergo telomere shortening and exhibit decreased telomerase activity with increasing time in vitro. Specific Aim 4: To determine whether microglial cells undergo telomere shortening and exhibit altered gene expression with aging in vivo. It is anticipated that results from the proposed studies will provide new insights into degenerative microglial cell changes that occur with aging. Preliminary studies suggest that microglial degeneration is much more pronounced in AD compared to non-demented individuals, and thus microglial dysfunction could be an important factor in AD pathogenesis.

The communication between neurons and microglia is bidirectional. Microglia modulate neurotransmission, facilitate synapse formation and dissolution, and provide neuronal protection, but cellular/molecular mechanisms are incompletely understood. Much of the premise for interactions between dopamine neurons and microglia is supported by presence of dopamine receptors on microglial cells allowing them to respond to neuronal signals. The idea is that dopamine receptor stimulation on microglial cells alters microglial function, which in turn could then (reciprocally) affect dopamine neurotransmission. Here we will use patch clamp electrophysiology, two-photon imaging, biochemical and histological approaches to determine whether and how depleting microglia affects dopamine neurotransmission and whether HIV-1 Tat, a protein produced in microglial cells following HIV-1 infection, disrupts dopamine neurotransmission by altering microglial/DA neurons interactions (Aim 1). We will examine how microglial activity is affected by dopaminergic signaling in the presence or absence of HIV- 1 Tat, and conversely how microglial products may modulate dopamine neurotransmission (Aim 2). Finally, since there is a high comorbidity between HIV-1 infection and drug abuse, and since both methamphetamine and HIV-1 Tat alter dopamine neurotransmission and affect the immune system, in Aim 3 we will determine how the combined exposure to HIV-1 Tat and methamphetamine influences these processes. The proposed work will address two significant knowledge gaps: 1) reveal the cellular/molecular mechanisms underlying bidirectional communication between dopamine neurons and microglia, and 2) determine how HIV-1 Tat modulation of this bidirectional communication reduces dopamine neurotransmission.

The central goal of this study is to investigate the functional and physical interplay between the dopamine (DA) D2 autoreceptor (D2R) and the dopamine transporter (DAT) and the role of this interplay in the regulation of DA neurotransmission. DA neurons are functionally heterogeneous with spatially separated somatodendritic and axonal projections initiating in two neighboring brain structures; ventral tegmental area (VTA) and substantia nigra (SN). Aberrations in DA neurotransmission are implicated in neuropsychiatric disorders; including schizophrenia, attention deficit/hyperactivity disorder (ADHD), drug addiction, and Parkinson's disease. Critical mechanisms in the regulation of DA availability at the synapse include the activation of D2 autoreceptors and DA uptake via DAT. Traditionally, these two mechanisms ? D2R and DAT- have been studied individually; however, exciting new evidence from in vitro experiments suggests that D2 autoreceptor and DAT interact physically and possibly functionally. Published data and our own preliminary findings support the hypothesis that D2 autoreceptor and DAT exist as a macromolecular complex and that D2 autoreceptor activation regulates DAT activity and trafficking in DA neurons through a GIRK-mediated mechanism. To address this hypothesis we will use a multidisciplinary approach combining molecular, biochemical, electrophysiological, and optic approaches in cultured DA neurons and brain slices containing somatodendritic regions of VTA, SNc and their projection areas (dorsal striatum and nucleus accumbens) where both DAT and D2R are co- expressed. In aim 1, we will use genetic, electrophysiological and optic tools to examine functionally whether D2R activation increases DAT activity and trafficking through a mechanism involving GIRK-mediated hyperpolarizarion of the cell membrane. In aim 2, we will use molecular, biochemical, and optic tools to examine the contribution of the D2R-DAT physical interaction to the D2R-mediated regulation of DAT activity and trafficking. We will compare and contrast findings obtained from SN and VTA, projections areas in the dorsal striatum vs nucleus accumbens, as well as samples from male and female animals. Given the role of D2R and DAT as therapeutic targets for DA-related conditions, the successful completion of this work will reveal the physiological significance of the interplay between presynaptic D2 receptor and DAT. The result of this work will have wide-ranging significance, as it will reveal a unique mechanism for the FDA approved D2R agonists' and DAT antagonists' regulation of dopamine neurotransmission.