Molecular Biology and Medicine A personal view

Molecular biology in medicine Understanding of normal and pathological processes in the body Understanding of drug action and development Genetics… … and personalised medicine?

Molecular biology and medicine My past research Career options

Investigation of the Neuronal Calcium Sensor Protein Neurocalcin d

Calcium signalling Ca2+ RyR Ca2+-CaM SERCA extracellular space LGC K+ VGC VGC PKC Ca2+ buffer SOC Ca2+ ATPase Ca2+ Ca2+ sensor NOS RyR Ca2+-CaM AC Na+/Ca2+ exchanger cAMP CaMK IP3R PDE Ca2+ buffer PKA ER SERCA Golgi CaMK mitochondrion nucleus

Calcium-myristoyl switch in recoverin

Proteins and the phospholipid bilayer

Inserting a gene into a recombinant DNA plasmid

Transforming E.coli with a recombinant DNA plasmid

Co-localisation of neurocalcin d and clathrin in HeLa cells overlay 20m

Summary and conclusions neurocalcin d binds actin, tubulin, clathrin HC and GAPDH this binding is Ca2+-dependent except for tubulin Ca2+-myristoyl switch operates in neurocalcin d in vivo Ca2+ induces neurocalcin d translocation to the TGN/perinuclear region neurocalcin d co-localises with clathrin in the TGN/perinuclear region at elevated Ca2+ this suggests one role of neurocalcin d may be in intracellular trafficking

Why should anyone care?? Certain neuronal calcium sensors have been implicated in aetiology of Alzheimer's disease (AD) Genome-wide association studies have since shown association of genetic variants of two of these proteins with AD+psychosis and late-onset AD One of these, VILIP-1, is being tested as a cerebrospinal fluid biomarker and a prognostic marker for cognitive decline in AD Braunwell K.H., Front Mol Neurosci. 2012; 5: 20. Kester M.I. et al, 2015, Alzheimer's Research & Therapy20157:59

Splicing Factors in Retinitis Pigmentosa

Retinitis pigmentosa (RP) progressive retinal degeneration incidence ~1 in 3500 begins in the periphery, results in narrowing of visual fields, night blindness, severe visual impairment, and blindness in ~1/3 of cases

Healthy and RP-affected eye

Proteins implicated in RP Phototransduction cascade components 6 Vitamin A transport/storage 6 Cell adhesion molecules 3 Intracellular transport 5 Transcription factors 3 Structural proteins 2 Splicing factors 4 Unknown 1

Pre-mRNA splicing

Ratio of spliced and unspliced RPGR RNA in control and splicing-factor mutant cell lines PRP31 PRP8 RPGR – autosomal dominant RP High level of alternative splicing in ex-in 15 Large number of disease causing mutations in this region

Summary and conclusions Splicing of RPGR is significantly reduced in PRP31 mutant cell lines

Obvious why we should care… "…an important role for pre-mRNA splicing in retinal homeostasis and the pathogenesis of retinal degenerative diseases. The development of novel therapeutic strategies to modulate aberrant splicing, including small molecule based therapies, has the potential to lead to the development of new treatments for retinal degenerative diseases." E.g. gene augmentation therapy (adding DNA containing a functional version of a mutated gene back into the cell, which produces sufficient amounts of a functioning protein to replace the defective/missing one): work started on PRP31. Liu M.M. and Zack D.J., 2013. Clin Genet. 2013 Aug; 84(2): 142–149. Hafler et al, 2016. Semin Ophthalmol. 2016;31(1-2):49-52. Beltran et al, 2015. Cold Spring Harb Perspect Med. 2015 Feb; 5(2).

Plasmalogen Function(s) in the Retina

Plasmalogens What is the function of plasmalogens in the retina? EPA DHA AA What is the function of plasmalogens in the retina?

How can we investigate this? DHAPAT -/- mice (DHAPAT catalyses 1st step in plasmalogen biosynthesis) reduce/abolish DHAPAT activity in retinoblastoma cells by RNA interference

RNA interference Y79 deficiency in DHAPAT/ADHAPS activity

Summary and conclusions

Omega-3 fatty acids https://www.healthline.com/nutrition/17-health-benefits-of- omega-3#section3 Anxiety & depression Eye health Brain health Cardiovascular health ADHD Autoimmune diseases Cancer Sleep Skin

Antioxidants

But Dr Go Ogle says it's a brain tumour.

Molecular biology in medicine Understanding of normal and pathological processes in the body Understanding of drug action and development Genetics… … and personalised medicine? Career options Clinical academic: "combine working as a specialist doctor with research and teaching responsibilities" (www.healthcareers.nhs.uk) https://www.studentdoctor.net/2014/02/04/academic-medicine-no-way-but-are-you-so-sure/

Career options

Career options

Be a doctor in your heart, and a scientist in your brain