Our laboratory is interested in elucidating the pathogenic mechanisms underlying neurological disorders that impact learning and memory. We are taking a multidisciplinary approach to investigate the molecular, cellular, and circuit basis of neurodevelopmental and neurodegenerative disorders.
The Role of Cdk5 in Neurodegeneration and Normal Brain Function
Alzheimer’s disease (AD) is a devastating and irreversible brain disorder that eventually leads to dementia and death. Cyclin-dependent kinase 5 (Cdk5) is a brain-specific protein serine/threonine kinase essential for brain development, synaptic plasticity, learning, and memory. We have shown that the hyperactivation of Cdk5 occurs when its regulatory protein p35 is cleaved by the Ca2+-activated protease calpain, under neurotoxic conditions, to liberate the carboxyl-terminal fragment p25. We hypothesized that p25 generation and accumulation play important roles in AD-like neurodegeneration. We created an inducible mouse model of p25 accumulation (the CK-p25 mouse) that displays key pathological hallmarks of AD, including profound neuronal loss in the forebrain, increased β-amyloid (Ab) peptide production, tau pathology, and severe cognitive impairment. In this model, increased β-amyloid peptide levels are observed prior to neuronal loss; furthermore, reducing β-amyloid peptide production ameliorates memory deficits in the CK-p25 mouse model, indicating that this event operates synergistically with p25, leading to the manifestation of neurodegeneration and memory impairment.
To further decipher the role of p25 generation in neurodegeneration, we created a mouse “knock-in” model whereby endogenous p35 gene is replaced by a mutant p35 resistant to calpain cleavage (Δp35KI). Δp35KI mice show normal hippocampus dependent learning and memory and LTP. However, they exhibit impaired hippocampal long-term depression (LTD). Moreover, Δp35KI hippocampal neurons are resistant to Ab peptide induced synaptic depression. The 5XFAD model is a well-established AD mouse model exhibiting abundant amyloid plaque pathology, inflammation and memory deficits by 6-month old. Interestingly, memory deficits are not observed in the Δp35KI/5XFAD compound mice, and the animals also show ameliorated inflammatory response and reduced Ab levels. These results strongly suggest that p25 mediates Ab peptide-associated pathology.
Currently, we are evaluating whether p25 generation also plays a role in Tau-mediated pathology. In addition, we are using Crispr/Cas-mediated genome editing to create calpain-resistant p35 alleles in human pluripotent stem cells (iPSCs) derived from AD patients (see below) to evaluate the role of p25 generation in the degeneration of human neurons and to create human cellular models of AD for therapeutic investigation.
Chromatin Remodeling and Learning and Memory
Among the available AD mouse models, the CK-p25 mouse uniquely recapitulates the profound neuronal loss that is observed in advanced stages of AD. Thus, we used this model to explore novel therapeutic approaches that may be beneficial for cognition even after profound synaptic loss and neuronal death have occurred. We show that treating CK-p25 mice with chemical histone deacetylase (HDAC) inhibitors induces robust synaptogenesis and dendritic growth, restores learning, and recovers long-term memory—even after massive neuronal loss has occurred. These findings demonstrate that an epigenetic mechanism involving increased histone acetylation and chromatin remodeling can be beneficial for learning and memory, and that these manipulations are effective even in the face of neuronal loss and neurodegeneration. These observations suggest that memory is not completely erased after neurodegeneration, and provide compelling evidence for developing HDAC inhibitors to reverse cognitive impairment in Alzheimer’s disease.
Specifically, our work has shown that the inhibition of HDAC2 by HDAC inhibitors is beneficial to learning and memory in both normal mice and in mouse models of neurodegeneration. We find that HDAC2 levels are increased in mouse AD models of as well as in postmortem brain samples from AD patients. More importantly, we show that the normalization of HDAC2 levels in mouse models of AD restores cognitive function, even following severe neurodegeneration. These findings advocate for the development of selective inhibitors of HDAC2 and suggest that cognitive capacities following neurodegeneration are not entirely lost, but merely impaired by this epigenetic blockade. Our current work is directed towards better understanding the HDAC2-corepressor complexes that are relevant to memory and which may be altered in AD.
To better understand the changes in the epigenome during neurodegeneration, we performed RNA- and chromatin immunoprecipitation (ChIP)-sequencing in the hippocampi of CK-p25 mice using seven different chromatin marks specific for active and inactive regulatory elements and gene bodies. We found that neuronal genes, those involved in synaptic transmission and learning/memory, are significantly downregulated, while innate and adaptive immune response genes are massively upregulated in the brains of CK-p25 mice. Notably, the direction of gene expression changes in the CK-p25 mice is remarkably similar to that observed in profiling studies conducted in the human AD hippocampus. From human data in which we examined genome regions orthologous to those containing disease-associated changes in enhancer elements in the mouse CK-p25 brain, we have identified master regulators of many genes that change expression in AD. Finally, we found that AD-associated genetic variants are enriched in the increased enhancer orthologs with a specific consistent temporal profile, and depleted in the decreased activity orthologs, implicating that alterations in the regulation of immune genes may underlie AD predisposition, while changes in neural pathways represent non-genetic effects. Currently, we are performing cell type specific epigenomic analysis and single cell RNA-sequencing in mouse and human brains to further understand the roles of different neural cell types including neurons, astrocytes, and microglia in AD-related neurodegeneration.
Neuronal Networks, Optogenetics, and Neurodegeneration
To better understand how disease genes and/or pathology impact cognitive function in disorders such as AD and autism, we have aspired to alter the activity of specific neuronal circuits and to evaluate the consequences on pathology, network activity, and behavior. The use of optogenetics allows for the manipulation of specific populations of neural cells in the brain. We have examined the effect of the stimulation or inhibition of specific cells within hippocampal, basal forebrain, and amygdalar circuits upon neurodegeneration and cognitive deficits. We continue to probe potential network dysfunction associated with neurodegeneration and other brain disorders, and to elucidate the contribution of various brain circuits in the early stage of pathogenesis in AD. In addition to the manipulation of specific circuits, we wish to map out which neural circuits are first disrupted by the deposition of Ab peptides and aggregated Tau protein in AD, and how Ab and Tau pathology propagates throughout the brain.
Current work also involves mapping various AD related pathology in a 3-D manner in mouse and human brains. We are using improved CLARITY techniques to interrogate the relationship of b-amyloid plaques, Tau tangles, neural inflammation and vasculature pathology in 3 different mouse models of AD and in postmortem human brain samples of AD subjects. We will also use this approach to assess how environmental enrichment and HDACi treatment, previously shown to improve cognitive function of AD mouse models, influence neuronal circuitry necessary for memory formation and retrieval.
We previously demonstrated that the disrupted in schizophrenia 1 (DISC1) protein, the product of a gene whose translocation strongly increases the risk for mental illnesses in a large Scottish pedigree, regulates neural progenitor proliferation by directly binding to and inhibiting GSK3β to modulate canonical Wnt signaling. Current work in the lab is elucidating the biological functions for genes that have been identified as either schizophrenia or autism risk genes, or both, in the development of the brain. For example, we have validated several mRNA targets for the microRNA MIR137 gene, which has been associated with a risk for schizophrenia and for autism in genome-wide association studies. Using induced human neurons (iNs) from more than 20 individuals, we investigated a haplotype (rs1006737) located within an intron region of the voltage gated L-type calcium channel subunit gene CACNA1C, significantly associated with risk for bipolar disease, autism, and schizophrenia. Whole cell patch clamp recordings and quantitative PCR experiments demonstrated increased L-type channel current density as well as increased mRNA expression of CACNA1C in iNs homozygous for the risk genotype compared with non-risk genotypes. These studies indicate that the risk genotype at rs1006737 is associated with significant functional alterations in human iNs and suggest that current and future drugs that interact with calcium channel signaling hold promise for these disorders.
Currently, our neurodevelopmental research program is aimed towards investigating autism spectrum disorder (ASD) risk genes, especially those implicated in chromatin remodeling and epigenetic regulation of gene expression. CHD8 is a bromodomain containing chromatin remodeler. In addition to its role at the chromatin, CHD8 has been shown to interact with the Wnt pathway, which is also heavily implicated in ASD phenotypes. Multiple de novo CHD8 loss of function mutations (termination, frame shift) have been identified in ASD subjects. We would like to use CHD8 as a mean to understand the importance of chromatin remodeling and epigenetic gene regulation in brain development and how dysregulation of this process leads to severe mental illnesses such as ASD. As part of the Library of Integrated Network-based Cellular Signatures (LINCS) project headed by Jacob Jaffe at the Broad Institute, we are systemically perturbing dozens of ASD risk genes in human neural cells and conducting in-depth proteomic profiling to identify the cellular pathways that link these genes to autism-like phenotypes.
Modeling neurological disorders using induced pluripotent stem cells (iPSCs) and tissue bioengineering
Our lab has collected human skin fibroblast lines from healthy individuals as well as late onset sporadic AD (LOAD), early onset familiar AD (fAD), ASD, schizophrenia, bipolar disease, and Down syndrome patients and reprogrammed them into induced pluripotent stem cells (iPSCs). We use genome-editing techniques such as Crispr/Cas9 to create isogenic cell lines to facilitate the assessment of phenotypic consequences of disease associated genetic variants. These iPSCs are then differentiated into excitatory neurons, astrocytes, and microglia that can be used for a number of basic and applied research purposes. For example, we can use this system to both examine how specific gene perturbations affect AD-like pathology directly in human neurons and glia, while at the same time screening libraries of drug-like chemicals in a high-throughput fashion to determine potential therapeutic candidates. Increasingly sophisticated culture techniques also allow us to evaluate how disease pathology and genetic variants affect each of the different cell types populating the brain. Using techniques of bioengineering combined with multiphoton deep imaging, optogenetics, and electrophysiology, we can recapitulate and study complex human brain tissue. In these “mini-brain” or organoid cultures, we can examine neuronal and glial activity and examine relevant disease phenotypes such as protein aggregation, neuronal connectivity, and synapse loss. Current efforts are further increasing the complexity of our three-dimensional culture systems to add engineered vasculature that will mimic the blood brain barrier. Ultimately, we hope that these techniques will facilitate drug discovery and testing by allowing us to directly screen engineered human brain organoids for compounds and therapies likely to work in the in vivo human brain.