Wishart Laboratory : Infantile Batten Disease – A synaptic Study

Maica Llavero Hurtado1, Tom Gillingwater1, Giusy Pennetta1, Jon Cooper2 & Tom Wishart1 1 University of Edinburgh, 2 Kings College London, United Kingdom. Contact: T.M.Wishart@ed.ac.uk (Neurobiology Division, The Roslin Institute, University of Edinburgh)

Informational Text Here

Acknowledgements: Dundee Proteomics Facility, Members of the Pennetta, Cooper and Wishart labs. Maica Llavero is funded by the Darwin Trust, Tom Wishart is funded by the BBSRC and MRC.

1. What is the problem?

DNA  muta)on  Non-­‐func)onal  CLN1  protein  

Neuron  degenera)on  

2. Why are synapses important?

…a  cell  body  

…an  axon  

…and  synapses  

A  neuron  has…  

5. Identification of protein differences in synapses

B  Thalamus  &  Cortex  dissec:on  

Isola:on  of  synapses    

A  

1 month 3 month 5 month 7 month

Late-symptomatic Early-symptomatic

Disease  onset?  

controls   controls   controls   controls  cln1/-­‐   cln1-­‐/-­‐   cln1-­‐/-­‐   cln1-­‐/-­‐  

Protein  extrac:on      

C   Protein  iden:fica:on  and  quan:fica:on      

D  

Protein  A   Protein  B  

6. Identification of disease regulators in other animal models

Healthy  eye  

Healthy  Fruit  Fly   BaGen  Disease  Fruit  Fly  

Small  and  degenera:ve  eye  

Healthy  synapse   Disrupted  synapse  

Protein  A   Protein  B  

We  can  modify  the  levels  of  our  protein  candidates  (iden:fied  in  5)  in  a  fly  model  of  BaGen  Disease  to  see  if  we  can  change  disease  

progression.  

Modifying  Protein  A  levels  we  make  the  eye  bigger.  

Makes  disease  beGer.  

Modifying  Protein  B  levels  we  make  the  eye  smaller.  

Makes  disease  worse.  

7. What does this means for therapy?

Biochemistry  techniques  

Small  animal  models  

Large  animal  models  

Human  

Can  we  target  our  candidates  with  drugs?  

Can  these  drugs  become  a  therapy  for  neurodegenera:on?  

The brain is a very complex organ. It contains billions of cells called neurons. Neurons form a very tight network of connections. When this network is disrupted it can cause a wide range of different diseases.

In Batten disease, synapses (communication points between nerve cells/neurons) begin to break down early in disease progression. The reasons why synapses are so vulnerable is unknown.

Our laboratory is trying to work out what mechanisms govern the vulnerability of synapses and could therefore be important in regulating disease progression.

Neurons appear to be quite complicated cells. There are many types of neurons but they all have synapses. Synapses are e s s e n t i a l c o n n e c t i o n s e n a b l i n g communication between neurons. Their stability is essential for normal brain function. Synapses break down at very early disease stages in many neurodegenerative cond i t i ons i nc lud ing A l zhe imer ’s , Huntington’s, Motor Neuron and Batten diseases. It is therefore imperative to understand the role of synapses in disease.

4. What happens in disease?

1 month 12 month 3 years 10 years

Late-symptomatic Early-symptomatic

Disease  onset?  

•  re:nal  degenera:on  •  blindness  •  cogni:ve  and  motor  

deficits  

•  seizures  •  flat  EEG    

•  Premature  mortality  

   

1 month 3 month 5 month 7 month

Late-symptomatic Early-symptomatic

Disease  onset?  

synap:c  breakdown  beginning  in  thalamus  

•  Seizures  •  Death  

Mouse  Model  

Human  

synap:c  breakdown  beginning  in  

cortex  

Mouse  brain  

The brain can be subdivided into different areas by the cell types contained within and functions performed by that portion of the brain. Not all brain regions are affected at the same time in neurodegenerative disease. For example, in Alzheimer ’s hippocampus goes f i rst . In Huntington’s it is striatum. This is also true for Batten Disease. In the mouse models available for the disease, synaptic breakdown is first detected in thalamus and then later in cortex. This raises the question: what makes some synapses more vulnerable than others?

CLN1  

3. What is CLN1 doing in the synapse?

CLN1 protein is not specific to neurons. It is involved in the function of lysosomes which are part of the machinery for waste clearance present in every cell. If lysosomes and therefore CLN1 are present in all cell types why does its loss have such a profound effect on neurons, presenting as a neurodegenerative disease? The presence of CLN1 protein must therefore have different consequences in different tissues and be particularly important for the maintenance of neurons. We therefore need to know what CLN1 interacts with in neurons and their synapses.

We use what is known about synaptic breakdown in CLN1 mouse models to examine how the composition of synapses changes due to the loss of CLN1 and throughout disease progression. We need protein extractions from mouse brain, specialised equipment, powerful computers and complex software to create and analyze the data. This workflow allows us to infer what CLN1 interacts with in synapses (see 3 above), to identify proteins which could control the stability of synapses (see 4 above) and regulate disease progression (see 6 next).

E   Correla:on  

We can model Batten disease in flies. Flies also have synapses which break down with Batten like mutations. However, their most obvious effect are alterations in the eye. Whilst they are obviously not as complex as humans, they are useful for testing candidates for their potential in therapy. After identifying interesting protein candidates we target them in our Batten disease flies. By modifying the levels of our protein candidates (identified in 5) we see if they can make the Batten disease eye fly better. We can successfully identify proteins changed in synapses due to loss of CLN1 which can change the disease in Batten flies.

These results are extremely preliminary and testing of candidates is needed in more “biologically relevant” larger models before we can be sure they well be useful for humans. However, these findings are an important proof of principle because it means that by starting with what is happening in synapses we can now search for candidates which change the rate of degeneration in Batten. It also means that successful candidates may also be effective in a range of neurodegenerative diseases where synapse are early pathological targets.

We are here

Some of the generic schematics presented here are modified from the internet.