Grant reports

2013

Carrier-Mediated Delivery of Therapeutic Proteins into the Brain

Fragile X Syndrome (FXS) is the most common single-gene cause of autism and the most common form of inherited intellectual disability in boys. The disorder is characterized by a range of symptoms from characteristic hand movements to severe intellectual impairment. FXS is currently a disorder without a cure or meaningful treatments. FXS stems from the FMR1 gene that is normally functioning in neurons, but is switched off during development due to a mutation.  The result is that the protein (FMRP) is not produced in FXS patients, and this deficiency causes the symptoms of Fragile X. Although FMRP can be delivered into the bloodstream of FXS patients, there are two major roadblocks that prevent FMRP from doing its job. Firstly, proteins on their own do not cross the “blood-brain-barrier”, which is a normal mechanism that creates a barrier between brain tissues and circulating blood, and serves to protect the central nervous system. Secondly, even if they did, they do not cross cell membranes to access their targets inside neurons. Our proposal aims to overcome both of these hurdles at once. By fusing FMRP to a non-toxic variant of diphtheria toxin (DT), produced by a specific bacteria, we can deliver FMRP across the blood-brain-barrier and into neurons. DT is a highly sophisticated protein that binds to its receptor on the cells that make up the blood-brain-barrier and is transported across and into the brain. Once there, it binds to the same receptor on the surface of neurons and is taken up by the cell and deposited into the cytoplasm inside. We are proposing to take advantage of this delivery vehicle that nature has evolved by fusing FMRP onto a non-toxic DT variant. Experiments have shown that if FMRP is reintroduced into the brains of the Fragile X mouse model which is lacking FMRP, it alleviates cognitive impairment and reverses autism-related symptoms. If successful, this will be the first direct treatment for FXS patients, and possibly even a cure.

Development of kainite receptors (KARs) in a mouse model of Fragile X syndrome

 Kainic acid is a potent central nervous system stimulant. It is used as the prototype neuroexcitatory amino acid for the induction of seizures in experimental animals, and acts via specific kainate receptors (KARs) located in the cell membrane of brain cells. KARs are one of three major types of glutamate receptor in the brain and when they are activated by the appropriate neurotransmitter molecule, the receptor changes shape and allows potassium and sodium ions to pass through the cell membrane. The Daniels lab has noted that of the various types of glutamate receptors, KARs  are the only type that has not been investigated for a pathophysiological or therapeutic role in Fragile X syndrome. Studies of the Fmr1 knockout mouse (Fragile X mouse model) and also of humans with Fragile X syndrome point to deficits in function of Purkinje cells in a part of the brain known as the cerebellum. Using the Fmr1 mouse as a model system along with appropriate controls, this lab has proposed three objectives: 1. investigate a novel role for KARs in the development of Purkinje cell function 2. examine the perturbation of KARs function in Fragile X mice, and investigate whether blocking the activity of the other type of glutamate receptors (known as mGluR) is sufficient to counteract any deficit seen in KARs.  3. Finally, the Daniels lab will also determine if normal nerve cell function in Fragile X mice can be achieved by promoting KARs activity through the use of drugs or chemicals that activate KARs.

Impaired cortical late-phase LTP and kainate receptor function in the Fmr1 mice

This lab is currently focused on PKMζ, a key molecule involved in long-term learning and memory. The symptoms of Fragile X syndrome are known to be caused by the absence of a single protein, known as FMRP. Dr. Zhuo’s lab has found that the upregulation of PKMζ by Forskolin treatment was blocked in the Fragile X mouse, indicating that FMRP may be critical in the regulation of PKMζ. More importantly, this lab has found that the Kainate receptor current and the number of these receptors present at the neuronal synapses are significantly reduced in the Fragile X mice. Further analysis showed that the movement of Kainate receptors within the cell was abnormal in the cultured cortical neurons of the Fragile X mice.

Kainate receptors have been found to play an important role in autism, and the results obtained in this lab strongly suggest that Kainate receptors also play a critical role in the pathology of  Fragile X syndrome.  Based on these observations, this lab plans to systematically study how PKMζ and the KA receptors are regulated by FMRP, and then find ways to “rescue” the L-LTP impairment and Kainate

The rescue of FMRP-related long term potentiation, learning and memory deficit in Frm1 knockout mice

Fragile X syndrome is caused by a mutation of the FMR1 gene, causing it to “shut down” and stop producing a key protein in the brain, called “FMRP”.  It is the absence of FMRP in the nerve cells of the brain that cause the symptoms of fragile X.

This lab will systematically investigate several approaches for treating FMRP-related learning and memory deficits seen in Fragile X mice (FMR1 KO mice). They have demonstrated that FMR1 KO mice have an impairment of synaptic strength associated with L-LTP(Late Phase Long-term Potentiation) and occurs in a part of the brain which is correlated with behavioural relevant memory functions.  The drugs MPEP and SB415286 may reduce the deficit of L-LTP in this part of the brain, known as the Anterior Cingulate Cortex (ACC). The drug, Rolipram may also be a potential candidate for treatment of learning and memory deficits in Fmr1 mice.  This lab will also study why the Fragile X mice have a deficit in the pre-LTP (Pre-Longterm Potentiation) response in their synapses, and look for ways to reverse the deficit.