Neurobiology

Zdenek Berger, Ph.D.

Elucidating Active Conformations of LRRK2 and Regulations of its Kinase Activity

(Advisor: Matt LaVoie, Dept. Of Neurology, Brigham and Women's Hospital, Harvard Medical School)

Parkinson’s disease (PD) is a devastating neurodegenerative disorder that affects 1-3% of the population over the age of 65. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of PD – they can be responsible for up to 40% of all PD cases in some populations, although the global frequency is substantially lower. Patients with LRRK2 mutations present clinically with typical PD, suggesting that mutations in this protein are relevant to the pathogenic process of all PD cases.

LRRK2 is widely recognized to be a kinase, mediating the phosphorylation of as yet unknown protein substrates. It has been suggested that excess LRRK2 activity may cause disease. Using the support from the Lefler Foundation, I will characterize different forms of LRRK2 we have isolated and determine the biologically relevant conformation of this kinase.

I am also investigating what processes make one form of LRRK2 more active than another one using neuronal cell culture coupled with biochemistry, various separation methods, and in vitro kinase assays. These findings will inform us about LRRK2's function in the cell and its role in the pathogenic process. Such information may also be valuable to an effective design/search strategy for LRRK2 inhibitors as potential PD therapeutics.

Eloise Hudry, Ph.D.

Gene therapy strategies in Alzheimer’s disease through peripheral administration of Adeno-associated vector (AAV)

(Advisor: Bradley Hyman, Dept. of Neurology/Alzheimer Disease Research Unit, Massachusetts General Hospital, Harvard Medical School)

Alzheimer’s disease (AD), the most prevalent neurodegenerative disease in the elderly, is histologically characterized by an abnormal accumulation of amyloid-ß (Aß) peptides in the brain. Strategies aiming at lowering the production of these neurotoxic peptides or at increasing their degradation may be therapeutically relevant. In my project, cerebral endothelial cells will be used as Trojan horses to secrete anti-Aß antibodies as a new immunotherapeutic approach in Alzheimer’s disease; or to increase the physiological export pathway of Aß out of the brain via LRP1 (Low density lipoprotein receptor related protein 1) overexpression.

Physiologically, the blood-brain barrier (BBB) maintains brain homeostasis and protects it against toxic molecules. However, it also prevents the intake of therapeutic drugs from the peripheral compartment to the central nervous system. By genetically modifying the intrinsic properties of the cerebral endothelial cells, we would like to take advantage of the close proximity between the brain vasculature and neurons in order to increase the clearance of Aß peptides. To this end, we plan to use new adeno-associated vectors (AAV) that were modified to efficiently transduce the cerebral endothelium after being administrated in the systemic circulation.

Two different therapeutic strategies will be evaluated to increase the clearance/efflux of Aß peptides. First, we will use these new AAV vectors to secrete single chain antibodies that specifically target Aß. Indeed, several passive immunization protocols have already demonstrated high potency to improve both histological and clinical features in AD mouse models, but a direct peripheral administration of IgG failed to efficiently cross the BBB and may result in complications such as vasogenic edema. We hope to avoid both problems by using modified cerebral endothelial cells as chronic suppliers of relatively low levels of anti-amyloid antibodies in the brain. Another promising strategy to clear Aß peptides from the brain is to increase their export into the vascular systemic compartment. Physiologically, it is now well established that the binding of Aß to LRP1 is the first step of Aß transcytosis into peripheral blood. We therefore plan to overexpress this receptor in the brain endothelial cells to increase the physiological efflux of these neurotoxic species out of the brain.

In this project, the use of peripherally administered vectors directed to the CNS capillary beds could offer a non-invasive way to deliver therapeutic molecules, exposing the entire brain to a locally synthesized reagent. By overexpressing Aß single chain antibodies or LRP1, we will test two promising approaches potentially able to affect the progression of the disease.

Xiaojin Liu, Ph.D.

Development of Neural Stem Cell (NSC) Therapy from A Novel Source of Autologous NSC, Filum Terminale

(Advisor: David Cardozo, Dept. Neurobiology, Harvard Medical School)

The goal of this project is to develop a novel approach to neural stem cell (NSC) therapy through isolation of neural stem cells from filum terminale (FT), an area of the central nervous system that has never been investigated for the presence of stem cells. Neural stem cells have previously been isolated from central nervous system (CNS) regions that are essential to normal brain function and difficult to access surgically. The FT is a vestigial structure at the caudal end of the spinal cord which is surgically easily accessible and which plays no functional role in the postnatal nervous system. It is a histologically primitive structure that represents the remnant of the developing spinal cord which provided innervation to the vestigial tail during early embryogenesis. These properties make it a potential source for autologous NSCs for therapeutic use. This project seeks to isolate NSCs from FT and to study their properties and therapeutic potential. FT will be obtained from postnatal rats. The tissue will be dissociated and neurospheres will be isolated and grown under conditions that promote stem cell survival. FT-NSC lines will be established and maintained. Neurospheres derived from FT will be examined for the expression of the NSC markers. FT-NSCs will be cultured in vitro under conditions that promote differentiation. Differentiated cell types will be identified using both immunocytochemical techniques and electrophysiological methods. Rat FT-NSCs will be transplanted into the CNS of either normal rodents or rodents that model spinal cord trauma or neurological disease. These experiments will measure the ability of transplanted cells to integrate into the host nervous system and to promote functional recovery in trauma and disease models. Preliminary results in the lab have demonstrated that neurospheres can be isolated and expanded from FT. FT-NSCs are positive for the NSC markers using immunocytochemical techniques.

The next step is to examine whether FT-NSCs have any beneficial effects in animal models of neurological diseases, such as spinal cord trauma, by transplanting FT-NSCs into CNS of the targeted animal models. By using FT-NSCs isolated from green fluorescent protein (GFP) transgenic rats, or generically, green rats, we will be able to track the transplanted cells and examine its integration and differentiation in vivo using fluorescent microcopy techniques.

Seth Margolis, Ph.D.

High Resolution Imaging of Molecular Pathways that Mediate Synapse Restriction

(Advisor: Michael Greenberg, Dept. Neurobiology, Harvard Medical School)

During normal brain development an intricate series of molecular events give rise to the development and maturation of excitatory synapses. The initial formation of excitatory synapses occurs when a developing axonal growth cone interacts with filopodia on the developing dendrite. This early step in synapse development is mediated by cell surface proteins (e.g. Eph/Ephrin, Neuroligin/Neurexin etc.) and appears to be independent of the release of neurotransmitter. Subsequent steps in synapse maturation occur in concert with input from the environment and are thus triggered by the release of glutamate at the developing synapse. These activity-dependent steps in synapse development include the maturation of dendritic spines, protrusions of the dendrites where the pre-synaptic terminals contact the dendrite to form excitatory synapses.

A second activity-dependent step in excitatory synapse development is the process of synapse elimination. In many neuronal circuits more excitatory synapses form than are maintained in the adult organism. In response to neuronal activity some synapses are strengthened while weaker synapses are eliminated. The process of synapse elimination is crucial for the proper development of neuronal circuits. Key features of dendritic spine development that have not been previously characterized are the molecular events that restrict spine growth spatially and temporally.

It is reasonable to hypothesize that neurons have evolved mechanisms to keep the extent of spine growth in check, and to make sure that dendritic spines form at the right time and place during brain development. We have identified a new protein which antagonizes excitatory synapse development. We will determine the role of this protein in the temporal and spatial restriction of excitatory synapse development using live imaging techniques and in vivo mouse model system approaches.

Won-Jong Oh, Ph.D.

Role of Plexin-D1 in the cerebellar circuitry formation during development and maintenance in the adult

(Advisor: Chenghua Gu, Dept of Neurobiology, Harvard Medical School)

The proper functioning of the adult nervous system relies on the correct wiring of connections established during development and on their maintenance and plasticity in adulthood. The goal of this study is to uncover how a major motor control pathway, the cortico-pontine/IO/DCN-cerebellar pathway, is established and maintained. We are focusing on Sema3E and Plexin-D1, a novel ligand-receptor pair, which was previously shown to affect vascular guidance during development. In the preliminary gene expression analysis in the nervous system, Plexin-D1 and Sema3E showed a striking expression pattern in the cortico-pontine/ inferior olive (IO)/ dorsal columen nuclei (DCN)-cerebellar pathway. Moreover, neuronal-specific Plexin-D1 knockout mice exhibit motor behavior impairment.

Therefore, we hypothesize that Plexin-D1 is required for the proper formation of the corticopontine/IO/DCN-cerebellar circuitry. In this study, my goal is to focus on the later part of the circuitry to elucidate the function of Plexin-D1/Sema3E in cerebellar circuitry formation and maintenance.

Yue Yang

NeuroD2 Regulation of Presynaptic Development

(Advisor: Azad Bonni, Dept. Pathology, Harvard Medical School)

Synapses represent the primary site of information transfer in the brain. Presynaptic axonal differentiation is an essential step in synapse formation and remodeling. Abnormalities of synapse morphology are thought to contribute to the pathogenesis of neurodegenerative diseases. Therefore, elucidation of the mechanisms that govern presynaptic differentiation is essential for a better understanding of brain development and disease.

I have discovered that the transcription factor NeuroD2 suppresses presynaptic differentiation in granule neurons of the rat cerebellar cortex in vitro and in vivo. I have also found that blockade of voltage-gated calcium channels (VGCCs) leads to NeuroD2 protein degradation and consequently increases the number of presynaptic sites in granule neurons.

Future questions include how does activity blockade trigger NeuroD2 degradation and what are mechanisms by which NeuroD2 suppress presynaptic development?

Long-Jun Wu, Ph.D.

Contribution of microglial voltage-gated proton channel, HV1, in NADPH oxidase-induced neuronal death in a mouse model of Alzheimer’s disease

(Advisor: David Clapham Lab, Dept. Cardiology, Children's Hospital Boston and Dept. of Neurobiology, Harvard Medical School)

Alzheimer’s disease (AD) is characterized by progressive cognitive impairment and profound neuronal loss. Microglia, the resident macrophages in the brain, are strongly implicated in the pathology of AD. In the AD brain, reactive microglia surround senile plaques and produce oxidative injury to neurons by releasing reactive oxygen species (ROS). The primary source of ROS and oxidative damage in the AD brain is derived from microglial NADPH oxidase. Microglial cells are depolarized and protons accumulate inside cells during NADPH oxidase activation. As excessive depolarization inhibits NADPH oxidase function, a charge-compensating mechanism is required to limit depolarization. The voltage-gated proton channel, HV1, is highly expressed in brain microglia and is ideally suited to this function because it is sensitive to both voltage and pH gradients. We hypothesize that microglial HV1 contributes to Aβ-induced NADPH oxidase activation, ROS production and neuronal death in a mouse model of AD. We established that HV1 is the molecular identity for voltage-gated proton currents, and generated HV1 knockout mice.

Taking advantage of these mice, we will address the following questions to test the hypothesis of HV1’s role in AD progression: (1) Determine whether voltage-gated proton currents exist in microglia and are mediated by HV1 in situ; (2) Test the role of HV1 in charge compensation for NADPH oxidase activation by Aβ and ROS production in microglia in situ; (3) Determine HV1’s contribution to ROS production and neuronal death in a mouse model of Alzheimer’s disease. The current study will be the first attempt to investigate the function of HV1 in brain, with the aim of evaluating HV1 as a potential therapeutic target for the treatment of AD.