Fred “Rusty” Gage is a world leader in neurogenesis and president of the International Society for Stem Cell Research. He has developed novel cellular models of neurological diseases (schizophrenia, MS, autism, Alzheimer’s, Parkinson’s) using human induced pluripotent stem cells (iPSCs) derived from donor patients and differentiated to specific neuronal cell types. These models are useful for studying disease mechanisms, identifying targets, screening compounds and stratifying patients. They are more predictive of disease phenotypes than current models. Gage has also developed an in vitro myelination model using iPSCs to be used for screening of pharmacological compounds and to better understand diseases such as schizophrenia and Parkinson’s.
Technologies Available for Collaboration
NeuroscienceNeurological disease modeling – Fred Gage
Animal models of neuronal loss in diseases such as Parkinson’s – Martyn Goulding
The fundamental mechanisms that contribute to the cognitive and motor deficits that accompany neurodegenerative diseases are poorly understood, due in large part to the progressive nature of these diseases. Goulding’s lab studies the immediate and downstream effects of neuronal cell loss, such as ongoing changes to spared neurons or to other cell types that make up the circuit, and determines how they adapt to the loss of a particular neuronal cell type. By selectively ablating specific neuronal cell types, in a time- and spatially-dependent manner, one can perform “surgical” lesions and examine the resultant changes with much greater precision. This allows for a more precise evaluation of the attendant cellular, synaptic and physiological changes that occur in response to the initial lesion. He has used this approach to develop a model of Parkinson’s disease.
Novel molecular targets for treating schizophrenia – Kuo-Fen Lee
Identifying Astrocyte-Secreted Factors to Treat Autism Spectrum Disorders – Nicola Allen
Multiple types of autism spectrum disorders (ASD) give rise to similar cellular defects in the developing brain – this is despite ASD being caused by distinct sets of genetic mutations or arising sporadically. Cellular phenotypes include altered neuronal death, reduced neuronal dendritic arbor growth, and defects in neuronal synapse formation and function. The similar cellular phenotypes present across multiple ASDs suggests they may share common defects in the molecular mechanisms that regulate neuronal development, and raises the potential of identifying common pathways for therapeutic targeting and treatment of sporadic ASD. Many features of ASD are non-cell autonomous for neurons, and can be induced by astrocytes, the most abundant cell type in the brain. Astrocytes from ASD mouse models induce dysfunction in wild type (WT) neurons, and WT astrocytes can rescue much of the dysfunction of ASD neurons (Fig 1). Astrocytes have important roles in brain development by secreting factors that regulate neuronal survival, dendrite outgrowth, synapse formation, and neuronal network function. Our lab has extensive experience in identifying and characterizing astrocyte-secreted factors in normal brain development, and we are applying this knowledge to identify core alterations in astrocyte secretion responsible for defects found in multiple types of ASD.
We hypothesize that multiple forms of ASD share common defects in astrocyte-secretion of neuron-supportive factors, and that these defects impair neuronal maturation and comprise a novel target for therapeutic intervention in multiple forms of ASD.
Figure 1. Astrocytes contribute to the pathology of ASD. A. WT astrocytes (green) secrete factors that promote the development of healthy neurons (green): neuron has a large central cell body, extends many long thin dendritic processes, and receives numerous syaptic connections (black). B. When ASD astrocytes (red) are cultured with ASD neurons (red) neuronal development is impaired; the neuron has a smaller cell body, extends less dendritic processes which are thick and stunted, and receives less synaptic connections. C. WT astrocytes release factors that are sufficient to rescue the phenotype of ASD neurons. D. ASD astrocytes release factors that are sufficient to impair the development of WT neurons.
To test this hypothesis we have:
1) Developed a protocol to purify and culture astrocytes from the postnatal mouse brain, in serum-free conditions to prevent induction of inflammatory genes.
2) Determined that proteins secreted by these astrocytes are sufficient to maintain neuronal survival and process outgrowth in vitro
3) Used mass spectrometry to identify the full complement of astrocyte-secreted proteins, a list of ~2000
4) Used RNA sequencing to identify astrocyte-expressed genes, and whether gene expression correlates with protein level.
We are now applying these methods to astrocytes from multiple models of ASD, to identify differentially secreted proteins that are candidates for the negative impact of ASD astrocytes on WT neurons. These candidates will be tested for their ability to regulate neuronal survival, neuronal process outgrowth, and synapse formation using in vitro assays.
This analysis of astrocyte-secretion, both in health and in ASD, will provide the first comprehensive analysis of the astrocyte secretome and identify novel targets for future therapeutic targeting in ASD.
Repair of synaptic connections following injuries such as stroke – Nicola Allen
Astrocyte Regulation of Neuronal Synapses and Circuits – Nicola Allen
Synapses are essential points of information transfer within neuronal circuits, and the correct formation and maturation of synapses is necessary for the brain to function throughout life. Astrocytes are the most abundant cell type in the brain, and many synapses have an astrocyte process associated with them. Landmark studies in the developing brain have shown that astrocytes secrete signals that can induce the formation of new synapses, alter the strength of synapses or cause the elimination of existing synapses. However the identity of all of these signals, and their specific mode of action, are not known. Our lab is working to identify how astrocytes interact with and regulate neuronal synapses throughout life: in development of neuronal circuits, in adult synaptic plasticity, and in synapse loss in aging.Specific areas of research:
- Identification and characterization of astrocyte-secreted signals that regulate neuronal synapse number and strength during development
- Determining what controls the release of synaptogenic factors from astrocytes
- Identifying how alterations in astrocyte protein secretion contribute to autism spectrum disorder
- Investigating if astrocytes in the adult brain limit synaptic plasticity and inhibit learning
- Investigating if astrocytes in the aging brain contribute to age-related synapse loss
- Determining if secreted signals from young astrocytes can be used to repair synapses after injury
To achieve these goals we take a multi-disciplinary approach:
- In vitro cultures of purified neurons and purified astrocytes, isolated using immunopanning, to identify novel astrocyte-secreted synaptogenic molecules and to dissect out the signaling pathways they regulate in neurons
- RNAseq and mass spectrometry to identify the astrocyte-specific transcriptome and secretome, across development both in vitro and in vivo
- Astrocyte-specific knock out mouse models to determine how identified factors regulate synapse and circuit formation and function in vivo
- Approaches such as electrophysiology to assess synaptic strength and plasticity, immunohistochemistry to assess synapse number, and behavioral assays to assess learning
Optogenetics in the treatment of Parkinson’s disease – Xin Jin
Imaging of specific neuronal cell types in awake, behaving animals – Axel Nimmerjhan
In vitro model of myelination and MS – Fred Gage
Summary of myelination model:
Myelin is an electrically insulating material that forms a membrane layer, the myelin sheath, around the axon of a neuron. It is produced by oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system. Myelination increases speed of impulse propagation by saltatory conduction through uninsulated portions of axon called Nodes of Ranvier. Loss of myelin membrane, as occurs in many neurological disorders such as multiple sclerosis, leads to disruption of electrical communication between neurons and permanent neuronal functional deficits including loss of motor control and cognition impairment. Multiple sclerosis alone affects over 400,000 people in the United States and more than 2.1 million people worldwide.
The myelination process has mostly been studied with isolated primary cells, in postmortem tissues, or with animal models. All approaches are restricted by variability among individuals, by the intrinsic differences between animal and human models and by the limited number of cells and animals, making large-scale experiments difficult. Embryonic stem cells (ESCs) allow for a relatively uniform population of a large number of cells and induced pluripotent stem cells (iPSCs) offer an opportunity to compare healthy and patient cells directly. We have established an in vitro (in a dish) myelination model using ESC- and iPSC-derived oligodendrocytes and neurons. Our assay will contribute to basic understanding of myelin formation leading to refined treatment strategies. Due to the reproducibility and reliability, our proposed assay will be available for pharmacological compounds screening and adaptation by other research groups to test their hypotheses. Currently, we achieved successful myelin formation using mouse ESCs and we are already gaining knowledge on the mechanics of myelin wrapping.
OncologyA cell-based screen to identify therapeutic compounds for pancreatic cancer – Ronald Evans
SKIP and cancer therapy – Katherine Jones
ULK1 Inhibitors, in combination with mTOR inhibitors as a cytotoxic cancer therapy – Reuben Shaw
While the role of mTOR in cell growth is well known, its role in autophagy has only recently been identified. Autophagy is the cellular process by which cells break down intracellular proteins and organelles under conditions of stress, freeing up metabolic intermediates and promoting cell survival. Dr. Shaw’s work recently demonstrated that a complex controlling the initiation of autophagy, composed of a kinase called ULK1, is inactivated by mTOR (Egan et al., Science 2011). Thus, inhibiting mTOR will lead to activation of autophagy which is a major pathway promoting cell survival. The propensity of mTOR inhibitors to promote tumor cell survival via ULK1-dependent autophagy may help explain why rapalogs are primarily cytostatic, and only effective as disease stabilizers rather than for regression. Dr. Shaw has postulated that combining inhibition of ULK1 with rapamycin treatment might convert the standard cytostatic effect of mTOR inhibitors into a cytotoxic effect, once the survival benefit of ULK1-initated autophagy is removed. His laboratory has developed a variety of inducible, genetically-engineered mouse models that give biological readouts of treatment regimes in settings that are predictive of human cancer subtypes. He has also developed a vast array of in vitro assays to test biological hypotheses across all members of the relevant pathways, and to screen for ULK1 inhibitors. He is currently developing small molecule inhibitors with a collaborator.
Cancer models that replicate the characteristics of human disease – Inder Verma
New targets for triple negative breast cancer – Geoff Wahl
Mouse models of non-small cell lung carcinoma – Reuben Shaw
MiscellaneousTherapy for hemophilia – Inder Verma
High throughtput screen for modulators of protein-protein interactions (PPI) – Geoff Wahl
Motor neuron dysfunction in ALS and spinal muscular atrophy (SMA) – Sam Pfaff