The Eye & Mitochondrial diseases Flashcards
1
Q
Neurotropic keratitis
A
Caused by damage to trigeminal nerve which results in damage in tear ducts, therefore decrease vitamins and GF into the cornea causing corneal epithelial breakdown and ulceration
- Cornea: neural crest origin
- Post mitotic tissue and does not replicate
2
Q
Retinal Detachment
A
- Not harmful in itself until it pulls the retina, but it causes floaters in vision.
- Caused by vitreous humour: losing volume by dehydration
3
Q
Wald’s visual cycle
A
- Photoreceptor pigment epithelium complex (PPE) is made up of RPE, smooth ER capping the photoreceptors
- The RPE has enzymes the turns all trans-retinal to 11-cis retinal. 11 cis retinal migrates to the photoreceptor where it interacts with opsin (cGMP) the photoreceptor and this interaction with light will turn it back to trans retinal and migrates back to RPE, this change is recognised by the cell and causes electrical simulation and the cycle begins again
- In the dark, cGMP levels are high and keep cGMP-gated sodium channels open allowing a steady inward current, called the dark current. This dark current keeps the cell depolarised at about -40 mV, leading to glutamate release which inhibits excitation of neurons
- Light closes cGMP-gated sodium channels, reducing the influx of both Na+ and Ca2+ ions. Stopping the influx of Na+ ions effectively switches off the dark current. Reducing this dark current causes the photoreceptor to hyperpolarise, which reduces glutamate release which thus reduces the inhibition of retinal nerves, leading to excitation of these nerves.
This reduced Ca2+ influx during photo transduction enables deactivation and recovery from photo transduction
4
Q
Glaucoma
A
- Glaucoma is a group of eye diseases which result in damage to the optic nerve and vision loss. Vision loss from glaucoma, once it has occurred, is permanent.
- The major risk factor for most glaucomas and the focus of treatment is increased intra-ocular pressure, problems with axoplasmic flow. Intra-ocular pressure is a function of production of liquid aqueous humour by the ciliary processes of the eye, and its drainage through the trabecular meshwork.
- Key findings: Cotton wool spots are an abnormal finding on funduscopic exam of the retina of the eye. They appear as fluffy white patches on the retina. They are caused by damage to nerve fibres and are a result of accumulations of axoplasmic material within the nerve fibre layer.
5
Q
Mitochondrial placement
A
All before the lamina cribosa where the nerve cells are unmyelinated. This assists in pushing along the signal propagation in the unmyelinated axon
6
Q
Properties of Mt
A
- 1200 proteins in the mitochondria
- 13 coded by mt DNA and the rest by nuclear chromosomes
- All 13 are involved in the electron transport chain
- It is a covalently closed DNA molecule located in the mitochondrial matrix with a heavy and light strand
- It high as high copy number inherited maternally
- It is vulnerable to ROS induced mutation
- Replication, transcription and translation is mediated by nuclear gene products
- There are 2 non-coding regions: The origins of H-strand (OH) and L-strand (OL)
- Highly organised with no introns and with no non-coding regions in processed transcripts
7
Q
Factors that affect penetrance of mt diseases
A
- Maternal inheritance
- Heteroplasmy
- Threshold effect:
* Many heteroplasmic mtDNA mutations are highly recessive, small amounts of wild type can protect against mutation effects. High levels of mutation can be tolerated by cell before it induces a respiratory chain defect. Threshold is different in different tissues and mutations.
- Threshold effect:
- Mitotic segregation:
* levels of mtDNA mutations can vary enormously between tissues. At mitosis both wild type and mutated mtDNA are randomly segregated to each daughter cell and undergo selection and amplification. This will affect both disease expression and inheritance - consider transmission mtDNA and prenatal testing) mtDNA segregation during maternal transmission and early embryogenesis
- Mitotic segregation:
- Two major mechanisms affect mtDNA genotypes during oocyte development — purifying selection (A) and the mtDNA genetic bottleneck (B). This is followed by a second bottleneck in early embryogenesis (C). By eliminating oocytes containing deleterious mtDNA mutations, purifying selection reduces the possibility of severe mtDNA disease and favours the preservation of functional mtDNA. Little is known about the exact timing of this purifying selection, but one study suggests that it occurs during oogenesis prior to the genetic bottleneck84. The mtDNA genetic bottleneck may occur due to reduction of mtDNA copy number or due to amplification of a subset of mtDNA molecules. Regardless of the mechanism, the result of the bottleneck is to decrease the number of haplotypes within an egg. This phenomenon also increases genotypic variance between mature eggs, a feature that facilitates the rapid segregation of mtDNA variants between generations. During very early embryonic development, mtDNA replication is not active, and the mtDNA content per cell is diluted due to an increase in cell number. It has been proposed that this reduction in mtDNA content per cell followed by the resumption of mtDNA replication results in a second bottleneck (C) during early embryonic development..
8
Q
Replication, transcription, translation
A
Replication
- No link at all to cell cycle and can replicate as and when is required
- Mitochondrial DNA is replicated by the DNA polymerase gamma complex (encoded by a nuclear gene)
- There are 2 models of replication in contentious debate:
- Asynchronous model: Replication starts in OH - primed by short processed RNA species, in one direction through a substantial part (e.g. two-thirds) of the circular molecule until you expose OL which initiates L-strand synthesis, and then replication of the light strand begins.
- Synchronous model: The more recently reported mode starts at a different origin within the D-loop region and uses coupled-strand replication with simultaneous synthesis of both strands
Transcription
- Strands are transcribed as single polycistrons
- Mitochondrial transcription is initiated from one of two promoters on the H-strand (HSP1 and HSP2) and from a single promoter on the L-strand (LSP).
- Transcription from HSP2, located close to the 5’ end of the MT-RNR1 gene, and from LSP, situated in the D-loop, generates almost genome length polycistronic transcripts, which are processed to produce the individual mRNA and tRNA molecules.
- HSP1 is located upstream of the MT-TF gene and produces only the two rRNAs, 12S and 16S, and the two mt-tRNAs, mt-tRNAPhe and mt-tRNAVal.
- The basal transcription machinery consists of a limited number of nuclear-encoded proteins: the mitochondrial RNA polymerase, the transcription activator, the transcription factor TFB2M, and the termination factor.
Translation:
- Nuclear DNA encoded proteins make factors for initiation, elongation and termination
- Mito-ribosomes are recycled in this process
- 80-85 nuclear DNA codes for mitochondrial chaperones
9
Q
Investigations
A
- clinical syndrome? e.g. MELAS etc.
- YES test for mtDNA or nuclear mutations in blood/urine (Can use urinary epithelial cells as it has a close correlation with muscle)
- Most diagnostic algorithms recommend evaluation of selected mitochondrial biomarkers in blood, urine, and spinal fluid. These typically include measurements of lactate and pyruvate in plasma and cerebrospinal fluid (CSF), plasma, urine, and CSF amino acids, plasma acylcarnitine, and urine organic acids
- next-generation sequencing (NGS) methodologies have emerged as the new gold standard methodology for mtDNA genome sequencing because they allow significantly improved reliability and sensitivity of mtDNA genome analyses for point mutations, low-level Heteroplasmy, and deletions, thereby providing a single test to accurately diagnose mtDNA disorders. This new approach may be considered as first-line testing for comprehensive analysis of the mitochondrial genome in blood, urine, or tissue, depending on symptom presentation and sample availability.
- Urine is increasingly recognized as a useful specimen for mtDNA genome analysis, given the high content of mtDNA in renal epithelial cells
- Most diagnostic algorithms recommend evaluation of selected mitochondrial biomarkers in blood, urine, and spinal fluid. These typically include measurements of lactate and pyruvate in plasma and cerebrospinal fluid (CSF), plasma, urine, and CSF amino acids, plasma acylcarnitine, and urine organic acids
- if NO, do muscle biopsy (sumit et al 2015 consensus from mitochondrial medicine society on the dx of mitochondrial disease)
- muscle biopsy: Histochemistry stain COX-SDH, Gomori trichrome (for ragged red fibers), Electron microscopy (EM) examines the mitochondria for inclusions and ultrastructural abnormalities
- biochemistry (measurement of chain complex activities and Ubiquinone -CoQ10):
- When obtaining a biopsy in the evaluation of mitochondrial disease, ETC enzymology (spectrophotometry) of complex I–IV activities in snap-frozen tissue or freshly isolated mitochondria should be obtained
- Defects in the synthesis of coenzyme Q10 (CoQ10) lead to a variety of potentially treatable mitochondrial diseases. CoQ10 levels can be measured directly in muscle, lymphocytes, and fibroblasts, although it is thought that the levels obtained in muscle are most sensitive for diagnosing primary CoQ10 deficiency.
- Depending on the type of mitochondrial disorder and type of central nervous system involvement, neuroimaging may or may not show structural alterations. Stroke-like lesions in a nonvascular distribution, diffuse white matter disease, and bilateral involvement of deep gray matter nuclei in the basal ganglia, mid-brain, or brainstem are all known classic findings in syndromic mitochondrial disease.
- YES test for mtDNA or nuclear mutations in blood/urine (Can use urinary epithelial cells as it has a close correlation with muscle)
10
Q
Optic nerve disease with disrupted mitochondrial function is caused by 3 main things
A
- Biochemical defect
- Impaired fusion
- mtDNA instability
11
Q
Biochemical Defect
A
LHON: Leber’s hereditary Optic Neuropathy
12
Q
LHON: Leber’s hereditary Optic Neuropathy
Clinical presentation
Genetics
Pathophysiology
Management
A
Clinical presentation
- An acute sequential neuropathy that begins with loss of central vision in one eye and progresses to visual loss in both.
- Presentation begins with caecocentral scotoma, central vision loss, and colour vision less. On fundoscopy acute swelling of the optic disk can be seen, telangiectasia vessels around optic disc, swelling of the retinal nerve fibre layer on visual field examination.
- 7-75 age of onset, however mostly seen at 20-30. 90% will get it by age 50
Genetics
- Maternally transmitted- 1o MtDNA disorder, however is not 100% penetrant
- 3 MtDNA point mutations account for 90% of cases:
- 1178G>A (60%)
- 14484 T>C (15%)
- 3406 G>A (15%)
- Part of Haplogroup J which gives 2x risk of visual loss
- 2 locus model: Recessive nuclear modifier gene on chromosome X. Xp21.1
- Acts in synergy with MtDNA and increased your susceptibility to visual loss
Pathophysiology
- Biochemical defect: All associated mutations are associated with a subunit in complex I, this impairs the function of the electron transport chain, increasing number of ROS produced while causing an energy deficiency in cells. Along with impaired glutamate transport, it causes apoptosis of RGC, optic nerve degeneration and visual failure.
- Although a globally problem, retinal ganglion cells are preferentially affected, reason for this are unknown, hypothesis infer that high energy demands of the cell elicit the cause.
Management
- Idebenone –
- reactive oxygen species scavenger and acts on the e- transport chain
- Increases the production of ATP
13
Q
Impaired fusion and mitochondrial instability
A
Dominant optic atrophy
14
Q
Dominant optic atrophy
Clinical presentation
Genetics
Pathophysiology
A
Clinical presentation
- It’s s an autosomally inherited disease that affects the optic nerves, causing reduced visual acuity and blindness beginning in childhood.
Genetics
- This condition is due to mitochondrial dysfunction mediating the death of optic nerve fibres. Retinal ganglion cells (RGCs) are preferentially affected in DOA.
-
Mt instability: OPA1 is linked to the formation of multiple somatic mtDNA deletions, and that these contribute to the phenotype. It is likely that OPA1 is a gene important for mtDNA maintenance, which should be considered in patients with unexplained multiple mtDNA deletions. Hudson et al 2008
- Alavi et al 2013, mtDNA deletions have been associated with OPA1 mutations
- Impaired fusion: It is the most common inherited optic neuropathy. Where 60% of cases harbour pathogenic OPA1 mutations. OPA1 has distinct roles in the fusion of mitochondrial inner membranes during mitochondrial fusion events, and in regulation of cell death. In DOA patients, 10% of muscle fibres were COX negative i.e. blue counterstain. The mosaic pattern in muscle is highly suggestive mitochondrial disease.
Pathophysiology
- The OPA1 gene a nuclear-encoded mitochondrial protein codes for a dynamin-related GTPase protein targeted to the mitochondrial inner membrane. It is involved in modelling cristae structures - where infolding in mt is held together by OPA1.
- Without OPA1, cristae loses its integrity
- Fission and fusion in mt is also regulated by OPA1. Mitochondria in normal cells have a lot of budding and fusion Biochemical and mitochondrial morphological studies on cells from patients affected by autosomal dominant optic atrophy have shown a severe defect in the shape (with a very remarkable fragmentation of the mitochondrial tubules in small spheres) and distribution of mitochondria, occurring independently from a bioenergetics defect (respiratory chain function, ATP synthesis, and reactive oxygen species production) or apoptosis, indicating that the mitochondrial fusion defect is the primary pathogenic mechanism
15
Q
Charcot Marie Tooth
Clincal presentation
Genetics & Pathophysiology
A
Clinical presentation
- A hereditary motor and sensory neuropathy causes axonal degeneration +/- demyelination and it is an inherited AD/AR/X-linked
Genetics/ Pathophysiology
- CMT type 2A - AD and the most common form with classic signs of optic neuropathy
- Caused by MFN-2 (nuclear genome) which is involved in mitochondrial fusion
- It is a GTPase protein in the mitochondrial outer membrane
- A small group of CMT-2A patients instead have the KIF1B (nuclear) mutation
- KIF1B protein is part of kinase 3 family that transports cargo along actin /myosin within axons and cells in general.
- It interacts with mitochondria and regulates their movement within the cell
22
Q
Lightowlers et al 2015
A
Pre-implant treatment:
Mitochondrial donation. Mitochondrial donation involves the transfer of the nuclear chromosomal DNA from an oocyte or zygote from a woman with pathogenic mtDNA mutation (shown in pink) into an enucleated, recipient donor oocyte or zygote (blue). In these techniques the mitochondria (and mitochondrial DNA) are from the donor (shown in green). (A) Metaphase II spindle transfer involves removal of the spindle from the donor egg and transfer of the patient’s spindle into the donor oocyte followed by fertilization and development. (B) Pronuclear transfer occurs after fertilization and the pronuclei are transferred from the patient zygote to the donor zygote
Pre-natal and Pre implantation testing
Prenatal testing at the chorionic villus stage or amniocentesis to determine the level of mtDNA mutation in the developing fetus. Preimplantation genetic diagnosis, determined on the basis of analysis of either one or two blastomeres of a day-3 embryo, is increasingly used to reduce the risk of transmitting some mtDNA mutations and has become an option for some women with specific mtDNA mutations (18,19). Furthermore, TALENS and other potential genetic editing techniques (see below) may be able to reduce the level of mtDNA mutation in individual oocytes (20). The latter is an attractive approach, but in cases with high levels of heteroplasmy, the total mtDNA copy number may be lowered to levels that prevent implantation, and these techniques would not be amenable for homoplasmic mtDNA mutations
