Wednesday, February 15, 2017

More answers, more questions

Today’s post is about my struggle to find answers to questions such as the following: 

  • Are the neurological issues seen in some cases of ND linked to the Blood Brain Barrier (BBB) leakiness of the cerebellum found in ND related research?
  • If yes, then why is it that some cases of ND have neurological issues while others do not?
  • Are the epilepsy & seizures reported in some cases of ND an outcome of ND?

I decided to get in touch with Prof. Jeremy Nathans, professor of molecular biology and genetics, of neuroscience, and of ophthalmology at the Johns Hopkins University School of Medicine, in order to try and make sense of all that I have read so far in relation to the above.

Before I present to you the ensuing conversation, here is a background to the questions posed by me:

From the research that I have read, it seems that 
  • there is a Blood Brain Barrier (BBB) dysfunction in the cerebellum and the olfactory bulb in ND (Nathans et al., 2014)
  • there is a reduction in blood vessel density in the cerebellum in ND (Luhmann et al., 2008
  • the gene Ndp is expressed in the cerebellum, hippocampus, olfactory bulb, cortex, and epithelium of the brain (Hartzer et al., 1999)
  • BBB dysfunction in the hippocampus is one of the many causes of epilepsy (van EA Vliet et al., 2014)

Further, traditionally the function of the cerebellum has been thought of as being motor control, but this view is being increasingly challenged. I cite a few examples:

  • Cerebellar function was once believed to be motor-specific, but newer findings suggest the cerebellum is also involved in higher-level brain processing. Functional imaging studies have also shown cerebellar activation in relation to language, attention, and mental imagery.  [Source: Boundless, 11 Oct. 2016. “Functions of the Cerebellum”  ]
  • The traditional teaching that the cerebellum is purely a motor control device no longer appears valid, if, indeed, ever it was. There is increasing recognition that the cerebellum contributes to cognitive processing and emotional control in addition to its role in motor coordination.[Source: Brain, Feb. 2006. “Cognition, emotion and the cerebellum”]

And so, on to the ‘conversation’ (which took place via email) with Prof. Nathans, presented here with his kind permission.

Qs: From the research done by you and your colleagues, would it be correct to infer that a leaky BBB is present in all cases of Norrie Disease (ND), and that the difference amongst various cases is primarily that of the extent of leakiness?

Ans: I do not think that there is any direct data on BBB integrity in humans with ND. Also, the BBB in most of the brain is protected from loss of Norrin by a second redundant signaling system that uses Wnt7a and Wnt7b (at least in mice). The retina does not have the back-up system (in mice).

Qs: While there is no direct data on BBB integrity on humans, I thought one could make inferences based on lab based research on animals?

Also, when you say "most of the brain", does this exclude the cerebellum & olfactory bulb? 

Ans: Extrapolating from mice to humans is usually OK, but not always. Yes, the cerebellum and olfactory bulb show a mild BBB defect when Norrin is absent - so for these two locations the BBB is not completed covered by the Wnt7a and Wnt7b system.

Qs: If one wanted to know if a similar BBB defect exists in the case of ND in humans, what would be the way to determine this? Would it show up in an MRI for example?

Ans: That is a very good question, and I do not know the answer. I think we would have to ask the neuro-radiologists.

Qs: I rephrase my very first question. From the research done by you and your colleagues, would it be correct to infer that a leaky BBB is present in the cerebellum and the olfactory bulb in all cases of a loss of function mutation in the Norrie gene (Ndp) in mice?

Ans: Phrased that way, the answer is yes. It is very consistent in mice.

Qs: Was there an opportunity to observe whether the extent of leakiness varied across different mutations of Ndp?

Ans: No, we only have one mouse mutation and it completely eliminates Norrin.

Qs: One of the symptoms of ND is neurological disorders. In ND related literature this is reported in up to 30-50% of cases. IF – and I realize this is a big if - the same BBB defects that are seen in Norrie knockout mice be found in patients of ND as well, then this could help explain these symptoms. Since not all cases of ND present these symptoms, I think it would be interesting to see if the extent of BBB leakiness varies across different Ndp mutations. Would you care to weigh in on this?

Ans: That is a very plausible idea.

Qs: There has been mention in ND related literature of epilepsy & seizures, and we see this in several cases of the present day online Norrie community also. 

Studies have shown that BBB dysfunction in the hippocampus is one of the many causes of epilepsy (van EA Vliet et al., 2014). 

While I could not find any mention of the hippocampus in your study, which used mice, I did find another, older study (Hartzer et al., 1999) which tells us that the gene Ndp is expressed in the hippocampus (& also the cortex!) of the rabbit brain (………… In order to further determine the role of the Norrie disease gene, we studied the distribution pattern of its mRNA in the retina and in brain by in situ hybridization. …….. High expression levels were also observed in the cerebellar granular layer, hippocampus, olfactory bulb, cortex, and epithelium of the rabbit brain. …….). 

So the logical conclusion would be that we cannot rule out the possibility that the gene Ndp may be expressed in the human hippocampus as well as the cortex? Would you agree?

Ans: The mouse data shows that Ndp is likely expressed widely (nearly everywhere) in the brain.

Qs: If Ndp is expressed widely in the brain, but it plays a role in BBB maintenance primarily in the cerebellum and olfactory bulb, then do we know what role it plays in the other parts of the brain?

Ans: It appears that signaling by a second system (based on proteins called Wnt7a and Wnt7b) functions in parallel to Norrin signaling in many parts of the brain (but not the retina) and in those regions it covers up the deficiencies that would be seen if Norrin alone were responsible. This is all based on work in mice.

Qs: A later study (Luhmann et al., 2008) speaks of reduced vessel density in the cerebellum of Ndph knockout mice. Is this consistent with your findings?

Ans: We agree. There is a subtle reduction in vessel density in cerebellum in Ndp KO mice.

Qs: Is the problem of how to restore the Norrin signaling in order to restore the BBB for ND patients under active consideration by your lab? If yes, then would this involve delivering the protein Norrin to the brain?

Ans: We have not pursued that line of investigation. Delivering therapeutic proteins to the brain is challenging because the BBB is a barrier to entry. Many biotech and pharmaceutical companies are working on this.

Qs: In speaking to another researcher, I had come to know that in the past it has been difficult to obtain a consistently good quality of Norrin - the quality varies over batches - something to do with the cystine knot structure of the protein. Would you know if this problem has been resolved or is this yet another hurdle to be overcome?

Ans: I have not tried using commercial Norrin protein, but in our own lab’s experience it is difficult to produce in large quantities. However, at least one research group appears to have solved this problem: Yvonne Jones at Oxford University has made large amounts of pure Norrin for structural studies.

I thank Prof. Nathans for his time and patience.

So where does this all take me?

I think the neurological issues seen in some cases of ND are related to the defects seen in the cerebellum of Norrie knockout mice.

I think the reason why some cases of ND present neurological issues while others do not, has something to do with the particular mutation – that is to say that it has to do with the extent of damage caused by a particular mutation.

I think that while there is no BBB defect seen in the hippocampus of Norrie knockout mice, the jury is still out on whether the epilepsy & seizures seen in some cases of ND are related to ND or not, because when I look at individual cases in our present day online Norrie community, in almost all - if not all – cases, the person who has experienced seizures, also presents neurological issues. I do not remember an instance where this is not the case. (The converse is not true – not everyone with neurological issues has experienced epilepsy, but this could once again be about the extent of damage caused by a particular mutation).  

There is more work to be done here.

Till next time then,

Meenu.