Browsing by Author "Feng, Guoping"

The development and function of the nervous system relies on complex regulation of gene expression programs. MicroRNAs (miRNAs) are small RNAs that have diverse functions in mammalian development and disease. In concert with the RNA-induced silencing complex, miRNAs repress translation by binding to target mRNAs. The nervous system contains the largest proportion of miRNAs, yet few have been functionally characterized in vivo.

miR-133b is a highly conserved miRNA embedded in the sequence of 7H4, a noncoding RNA that is enriched at the neuromuscular junction (NMJ), a large synapse that is essential for eliciting muscle contraction and movement. I have found that, like 7H4, miR-133b expression is enriched at the NMJ and upregulated postnatally, coinciding with important events in synaptic maturation, including synaptic growth and elimination. Knockdown of miR-133b in postnatal muscle by electroporation of modified antisense oligonucleotides gave rise to abnormally large synapses, indicating a role for miR-133b in synaptic maturation. To specifically remove miR-133b in vivo, I generated a mouse containing a targeted deletion of the miR-133b stemloop. NMJ maturation and synapse elimination proceeded normally in miR-133b knockout mice, suggesting that miR-133b may have other functions at the synapse. The expression of 7H4 and miR-133b is upregulated following nerve transection, consistent with a role in synaptic regeneration. Indeed, NMJ reinnervation is delayed in miR-133b KO mice following nerve crush, but not nerve cut. These data suggest that miR-133b may have a specific protective function at the synapse that could be relevant to disease states, including amyotrophic lateral sclerosis (ALS), where NMJ denervation occurs following motor neuron cell death. However, loss of miR-133b did not affect survival or disease progression in the SOD1(G93A) mouse model, differentiating its role from that of miR-206, another miRNA found in 7H4.

miR-133b has recently been proposed to regulate the development and maintenance of midbrain dopaminergic (mDA) neurons. mDA neurons have critical functions in the control of movement and emotion, and their degeneration leads to motor and cognitive defects in Parkinson's disease. miR-133b is enriched in the midbrain and regulates mDA neuron differentiation in vitro by targeting Pitx3, a transcription factor required for appropriate development of substantia nigra DA neurons. However, the function of miR-133b in the intact midbrain has not been determined. miR-133b KO mice have normal numbers of midbrain dopaminergic neurons during development and aging. Moreover, dopamine neurotransmitter levels are unchanged in the striatum and other brain regions, while expression of dopaminergic genes including Pitx3 is also unaffected. Finally, miR-133b null mice display normal motor coordination and activity, suggesting that miR-133b does not play a significant role in the development or maintenance of the mDA neuron population.

Obsessive-compulsive disorder (OCD) is a devastating illness that afflicts around 2% of the world's population with recurrent distressing thoughts (obsessions) and repetitive ritualistic behaviors (compulsions). While dysfunction at excitatory glutaminergic excitatory synapses leading to hyperactivity of the orbitofrontal cortex and head of the caudate - brain regions involved in reinforcement learning - are implicated in the pathology of OCD, clinical studies involving patients are unable to dissect the molecular mechanisms underlying this cortico-striatal circuitry defect. Since OCD is highly heritable, recent studies using mutant mouse models have shed light on the cellular pathology mediating OCD symptoms. These studies point toward a crucial role for deltaFosB, a persistent transcription factor that accumulates with chronic neuronal activity and is involved in various diseases of the striatum. Furthermore, elevated deltaFosB levels results in the transcriptional upregulation of Grin2b, which codes GluN2B, an N-methyl-D-aspartate glutamate receptor (NMDAR) subunit required for the formation and maintenance of silent synapses. Taken together, the current evidence indicates that deltaFosB-mediated expression of aberrant silent synapses in caudate medium spiny neurons (MSNs), in particular D1 dopamine-receptor expressing MSNs (D1 MSNs), mediates the defective cortico-striatal synaptic transmission that underlies compulsive behavior in OCD.

Developmental disabilities, including intellectual disability (ID), attention-deficit hyperactivity disorder (ADHD), and autism spectrum disorders (ASD), affect approximately 1 in 6 children in the United States. Attempts to produce treatment for developmental disabilities have been hampered by our current lack of understanding of the molecular mechanisms underlying these disorders. Advancements in genome sequencing and animal modeling technologies have proven to be an invaluable resource in the elucidation of potential disease mechanisms, with recent studies reporting novel mutations of the Ptchd1 and Shank3 genes in patients with developmental disabilities. Though these two genes have been proposed to play important roles in neural development, their function in the normal brain and defective behavioral output are poorly understood.

In this dissertation, I characterize the circuit and behavioral dysfunction of the genetically-engineered Ptchd1 and Shank3 knockout mice. With respect to Ptchd1, I found that expression is developmentally enriched in the thalamic reticular nucleus (TRN), which is a group of GABAergic neurons serving as the major source of inhibition for thalamo-cortical neurons. Slice and in vivo electrophysiological experiments revealed that deletion of this gene in mice disrupts SK2 currents and burst firing mechanisms in the TRN, a region that has previously been shown to play an important role in sleep, attention, and cognition. Consistent with clinical findings, Ptchd1 knockout mice display behavioral phenotypes indicative of hyperactivity, attention deficits, motor dysfunction, hyperaggression, and cognitive impairment. Interestingly, attention-like deficits and hyperactivity are rescued by SK2 pharmacological enhancement, suggesting a potential molecular target for developing treatment.

Shank3 knockout mice display ASD-like phenotypes, including social interaction deficits and repetitive behaviors. In addition, biochemical, electrophysiological, and morphological abnormalities were discovered in the medium spiny neurons (MSNs) of these mice. However, the exact neural circuits and cell types responsible for the autistic-like behaviors have not been identified. To address this important question, I developed a new conditional Shank3 knockout mouse. Importantly, the behavioral abnormalities reported in the original Shank3 knockout mice were recapitulated in this novel conditional Shank3 knockout mouse, indicating that this mouse may be useful for future pathway-specific dissections of ASD-like behaviors. Together, these two sets of studies not only provide mouse models for dissecting the function of PTCHD1and SHANK3 in normal and abnormal neural development, but also demonstrate a critical role for PTCHD1 in TRN neurons and SHANK3 in MSN cells and in the case of PTCHD1, identify potential cellular and circuit pathway targets for much-needed pharmacological intervention.