neuro
  • BACKGROUND
  • PROJECT #1
  • PROJECT #2
  • PROJECT #3
  • EXPERIMENTAL APPROACHES
  • VIDEO GALLERY

CNS DISEASES:

I) Transcriptional control of axon growth and regeneration

The molecular mechanisms that control the capacity of adult neurons to regenerate axons are an important unsolved question in neurobiology. Neurons of the mammalian CNS regenerate minimally after injury. Failure of CNS axon regeneration is attributed to an inhibitory environment and an age-dependent decline of intrinsic axon growth potential. In contrast to CNS neurons, injured neurons in the peripheral nervous system (PNS) can regenerate axons, but this requires both activation of a neuronal pro-regenerative transcription program and Schwann cell plasticity. Our laboratory has been studying the close collaboration between regeneration-associated transcription factors, such as Smad1, and epigenetic machinery that modifies chromatin landscape in adult neurons to activate a growth state. We also study molecular mechanisms underlying glial cell plasticity after injury. T hese insights will help to identify molecular targets to promote axon regeneration after CNS injury.


II) Neural stem cells and neural repair after stroke

Stroke recovery hinges on axonal sprouting of surviving neurons in stroke penumbra and plasticity of cortical circuits to reconstruct connectivity. Adult neural stem cells (NSCs) play an important role in modulating injury response after cerebral ischemia. In addition, transplantation of NSCs or iPSCs holds tremendous promise as cell replacement therapy. Our laboratory is interested in investigating novel molecular players in mediating axonal sprouting and cortical circuits plasticity after stroke. We employ a variety of tools that include mouse genetics, NSC biology, stroke models, imaging, and tissue-engineering techniques. III) Glioma stem cell biology Tumor stem cells are believed to be a special subpopulation that exhibit enhanced tumorigenic capacity and resistance to conventional therapy. T hey also are highly infiltrative. Our laboratory is interested in comparing and contrasting NSCs and glioma stem cells in their migratory, self-renewal, and proliferative behaviors and the underlying molecular mechanisms.

III) Glioma stem cell biology

Tumor stem cells are believed to be a special subpopulation that exhibit enhanced tumorigenic capacity and resistance to conventional therapy. T hey also are highly infiltrative. Our laboratory is interested in comparing and contrasting NSCs and glioma stem cells in their migratory, self-renewal, and proliferative behaviors and the underlying molecular mechanisms.

Project 1: To study whether Smad1 plays key roles in the developmental growth of axons and in the conditioning lesion paradigm in vivo.

We will take advantage of Smad1 conditional knockout (cko) mice by breeding them to neruo-specific Cre lines. We also plan to directly inject Adeno associated virus-Cre into mice to knock out Smad1 in selected cell populations. In addition, we have generated transgenic mice with doxycycline-inducible small-hairpin RNA (shRNA) to knock down Smad1. Using these mice, we will be able to manipulate BMP/Smad1 signaling in a temporally and spatially specific fashion. We will conduct in vitro cell culture neurite outgrowth assays and perform in vivo conditioning lesion by sciatic transection followed by spinal cord injury to assess whether the conditioning effect will be abolished without Smad1.

To study whether Smad1 plays similar roles in axon growth during development, we have first confirmed that Smad1 is expressed in embryonic DRG, commissural, and motor neurons at the time when they are actively extending axons. I plan to use the above mentioned Smad1 mice for in vivo studies. Specific Cre lines will be used to selectively knock out Smad1 in specific neuronal populations. This will be complemented by manipulation of the Smad1 signaling in vitro by siRNA or BMP stimulation in dissociated cell culture and in explants. Different classes of neurons will be studied.

Project 2: To study the mechanisms of how Smad1 is induced and phosphorylated after peripheral (but not central) axotomy, and to identify downstream targets of Smad1.

Our preliminary data showed that axotomy-triggered Smad1 activation occurs unexpectedly through a BMP-independent pathway, i.e. C-terminal phosphorylation of Smad1 occurs not through the BMP receptor serine-threonine kinases. We plan to identify novel kinase(s) that phosphorylate Smad1 and promote axon growth. Either a candidate approach or an unbiased screen of a kinase library will be used. The identified kinase will be further studied to assess whether its activation is specific to peripheral axotomy; is it associated with microtubules; where does it reside, in axons or in cell body; is retrograde signaling from injury site involved in its activation? A combination of primary cell culture, biochemical and molecular approaches, and imaging techniques will be used. To identify downstream targets of Smad1, either gene profiling or proteomic analysis followed by mass spectrometry comparing neurons from Smad1 knockout mice to wildtype neurons will be used. A more focused study to compare different phosphorylated downstream targets will also be performed by immunoprecipitation using antibodies against phospho-tyrosine or phospho-Ser/Thr followed by protein identification.

Project 3: To reconstruct the cascade of TF activation that rekindles the regenerative ability in adult neurons.

To study other TFs in axonal growth, their synergistic cooperation, and the sequence of activation (immediate early gene vs. late gene),  we intend to use two approaches: 1) candidate approach by examining the remaining 18 transcription factors identified from gene profiling of the preconditioned DRG neurons as mentioned above. We will use various manipulations such as siRNA, ko and cko, shRNA both in vitro and in vivo. For example, we have already generated Tg mice with inducible-shRNA against ATF3.  2) Genetic approach by transfecting neural cell lines with transposon or RNAi library of TFs to screen for novel regulators of neurite outgrowth. The validated TF or novel genes can then be further studied using murine axon injury models.

Experimental Approaches:

Our lab is trying to reconstruct the activation cascade of transcription factors and their downstream targets that controls the developing axon growth and rekindles the axon regenerative ability of adult neurons. The goal requires the integration of molecular biology, cell cultures based assays, biochemical techniques, imaging, mouse genetics and in vivo mouse models of axon and neuronal injury.

I) Dissociated neuronal cultures and explant cultures

We use dissociated neuronal cultures (e.g. DRG neurons, cerebellar granule cells) and explant cultures (DRG, commissural neuron explants) to conduct cell-based assays. A combination of adult, neonatal or embryonic cell cultures is used with gene manipulation techniques for gain- or loss- of function studies, such as siRNA transfection, electroporation of DNA plasmids or viral infection. Neurons from either wild type mice or genetically manipulated mice are used for comparison.

II) In vivo axonal and neuronal injury models

We validate our in vitro findings in vivo using mouse axon injury models. For peripheral nerve injury, we use sciatic nerve transection model. For CNS axon injury, thoracic spinal cord injury model is used. Either the ascending spinothalamic tract or the descending corticospinal tract can be traced by injecting appropriate dyes, followed by imaging analysis. For neuronal injury models, we are interested in establishing the classic stroke model, i.e. the middle cerebral artery occlusion (MCAO) model, which is reproducible in stroke size and location and has a well-defined border, which facilitates studying of neurogenesis at the stroke border.

III) Temporally and spatially specific gene manipulation in mice

We use a variety of techniques to manipulate molecular components of signaling pathways in a temporally and spatially specific fashion. We use the Cre-LoxP conditional knockout mice to knockout genes in specific population of neurons. We also plan to directly inject AAV-Cre into mice to knockout genes in selected neurons with precise temporal control. In addition, we have generated transgenic (Tg) mice with doxycycline-inducible small-hairpin RNA (shRNA) to knock down transcription factors. In future, the lab is also interested in using Piggybac transposon mutagenesis to conduct forward genetic screen for novel genes that control axon growth or novel pathways that contribute to CNS tumorigenesis.

 

 

 

zou lab
laboratory of axonal growth & neuronal regeneration