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The Biochemistry of Adrenoleukodystrophy (ALD) Paul Watkins MD, PhD Kennedy Krieger Institute & Johns Hopkins University Baltimore, MD.

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Presentation on theme: "The Biochemistry of Adrenoleukodystrophy (ALD) Paul Watkins MD, PhD Kennedy Krieger Institute & Johns Hopkins University Baltimore, MD."— Presentation transcript:

1 The Biochemistry of Adrenoleukodystrophy (ALD) Paul Watkins MD, PhD Kennedy Krieger Institute & Johns Hopkins University Baltimore, MD

2 Before we begin… ALD is a very complex disease, both clinically and biochemically ALD is a very complex disease, both clinically and biochemically We have known about the basic biochemical defect since the 1980s We have known about the basic biochemical defect since the 1980s The ALD gene was discovered in 1993 The ALD gene was discovered in 1993 but… Many questions remain unanswered Many questions remain unanswered

3 Part 1 Fatty acid biochemistry

4 Some basics… What is Biochemistry? What is Biochemistry? The chemistry of biological systems The chemistry of biological systems The chemistry of life The chemistry of life Need to know a little bit of biology... Need to know a little bit of biology... Need to know a little bit of chemistry... Need to know a little bit of chemistry...

5 Chemical Elements Living things are composed of several chemical building blocks, called “elements” Living things are composed of several chemical building blocks, called “elements” Other important elements include: Other important elements include:  Nitrogen (N)  Sulfur (S)  Phosphorus (P) The most abundant elements are: The most abundant elements are:  Carbon (C)  Hydrogen (H)  Oxygen (O)

6 Chemical Bonds Elements can link together by forming chemical “bonds” Elements can link together by forming chemical “bonds” There are rules that must be followed: There are rules that must be followed:  Carbon (C) can bond to 4 other elements  Hydrogen (H) can only make 1 bond  Oxygen (O) bonds to 2 other elements  Nitrogen (N) bonds to 3 other elements Substances containing elements linked together by chemical bonds are called “Compounds” Substances containing elements linked together by chemical bonds are called “Compounds”

7 Chemical compounds: some examples Water (H 2 O) H-O-H Water (H 2 O) H-O-H Ammonia (NH 3 ) Ammonia (NH 3 ) H-N-HH-N-HH-N-HH-N-HH Oxygen (O 2 ) O=O Oxygen (O 2 ) O=O In oxygen, there are 2 lines between the Os. This is a stronger “double bond” Methane (natural gas) (CH4)(CH4)(CH4)(CH4) H-C-HH-C-HH-C-HH-C-H HH

8 Biochemical Problem in ALD Too much Very Long Chain Fatty Acid (VLCFA) Too much Very Long Chain Fatty Acid (VLCFA) Buildup of VLCFA is in all parts of the body, especially: Buildup of VLCFA is in all parts of the body, especially:  Brain & spinal cord  Adrenal Glands VLCFA’s in blood plasma allows diagnosis of ALD by blood test

9 So what is a Fatty Acid? Made up of 3 chemical elements Made up of 3 chemical elements  Carbon (C)  Hydrogen (H)  Oxygen (O) A Fatty acid is also a chemical compound A Fatty acid is also a chemical compound

10 “Chains” of C and H form the “fatty” part “Chains” of C and H form the “fatty” part of a fatty acid: Fatty Acids Fatty Acids can have a chain that is... Fatty Acids can have a chain that is...  Short: 2-4 C’s  Medium: 6-10 C’s  Long: C’s  Very Long: 22-? C’s HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H H-H-

11 Fatty Acids C, H, and O bond together to form the C, H, and O bond together to form the “acid” part of a fatty acid: “acid” part of a fatty acid: O-C-O-H O-C-O-H Notice that while C is bonded to the O on Notice that while C is bonded to the O on the right by a single line (bond), it is the right by a single line (bond), it is bonded to the O above by 2 lines (a bonded to the O above by 2 lines (a “double bond”). “double bond”).

12 Fatty Acids OC-O-HOC-O-H H-H- HC-HHC-H Short-chain FA: Acetic acid (vinegar) HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H HC-HHC-H OC-O-HOC-O-H H-H- Long-chain FA: Palmitic acid (C16:0) Very long-chain FA: Cerotic acid (C26:0) (about 50% longer than long-chain FAs)

13 What does the body do with Fatty Acids? Like carbohydrates (sugars), fatty acids are used as a “fuel” for the body:  Breakdown of fatty acids  Energy + Heat + C, H, and O that are recycled Energy + Heat + C, H, and O that are recycled  The energy released from FA breakdown is stored as ATP, which is used to drive muscle contraction, heart pumping, breathing, brain function, and many other processes

14 Popular misconception: Fatty acid molecules (including VLCFA) are freely floating around in our bloodstream, cells, and organs 99% of fatty acids are found in the more complex molecules shown in the next slide Reality:

15 What does the body do with Fatty Acids? Fatty acids are used to make complex fats (“lipids”), and fatty acids are the “building blocks” Some examples of complex lipids: Triglycerides – the fat on a piece of meat (or on us!) Phospholipids – the molecules that make up the membranes that surround all our cells and tissues Glycolipids & Sphingolipids – molecules found in brain cells, especially in myelin membranes, that are necessary for normal brain functions

16 VLCFAs Like other fatty acids, VLCFAs are normal components of the body and are necessary for life. Like other fatty acids, VLCFAs are normal components of the body and are necessary for life. VLCFAs – important part of brain cell membrane lipids, especially myelin VLCFAs – important part of brain cell membrane lipids, especially myelin

17 VLCFAs – where do they come from? VLCFAs come from: VLCFAs come from:  Diet  Elongation of shorter fatty acids (= “synthesis”) (= “synthesis”)

18 VLCFAs (contd.) The balance between “just enough” and “too little” or “too much” The balance between “just enough” and “too little” or “too much” Intake + synthesis Excretion + breakdown too little too much

19 VLCFAs (contd.) Likely causes of excess VLCFA in ALD: Likely causes of excess VLCFA in ALD:  Body makes too much  Body cannot remove excess Early studies showed that the process which Early studies showed that the process which degrades VLCFAs (“  -oxidation”) was defective degrades VLCFAs (“  -oxidation”) was defective in cells from ALD patients in cells from ALD patients Intake + synthesis Excretion + breakdown too much too little

20 Fatty acid break- down (  -oxidation): removal of 2 carbons at a time * * * * * * * Four chemical reactions * O-H

21 VLCFAs (contd.) More recent studies indicate that VLCFA More recent studies indicate that VLCFA synthesis (elongation of long-chain FAs, or synthesis (elongation of long-chain FAs, or LCFA) is also increased in cells from ALD LCFA) is also increased in cells from ALD patients patients Intake + SYNTHESIS Excretion + breakdown too much too little

22 VLCFA Synthesis C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-OC-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-O C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-OC-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-O C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-OC-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-O C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-OC-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-O C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-OC-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-O C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-OC-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-O O O O O O O Dietary LCFAs

23 FA activation To do either process – break down VLCFA or make them from LCFA – the FAs first need to be “activated” To do either process – break down VLCFA or make them from LCFA – the FAs first need to be “activated” Activation means attaching the FA to a helper molecule called “Coenzyme A” (or CoA for short) Activation means attaching the FA to a helper molecule called “Coenzyme A” (or CoA for short) We would write this as “LCFA-CoA” or “VLCFA-CoA” We would write this as “LCFA-CoA” or “VLCFA-CoA” (Remember this when we get to Part 2!) (Remember this when we get to Part 2!)

24 A brief summary… Fatty acids are chemical compounds made of C, H, and O Fatty acids are chemical compounds made of C, H, and O VLCFA are ~50% longer than LCFA that are abundant in our normal diet VLCFA are ~50% longer than LCFA that are abundant in our normal diet The elevated VLCFA levels in ALD come from both increased synthesis and decreased degradation Unanswered questions… Which is more important? Which is more important? How does any of this relate to the ALD gene and protein? How does any of this relate to the ALD gene and protein?

25 Part 2 Genes and Proteins

26 Genes and proteins All ALD patients have a “mutation” in their All ALD patients have a “mutation” in their ALD gene ALD gene What does that mean???? What does that mean????

27 A view through the microscope… Cell Nucleus Chromosomes

28

29 The X-Chromosome The ALD gene is found at the tip of the longer arm (“q”) of the X- chromosome p q

30 The “Double helix” of DNA is made up of 4 chemicals called “bases”: Adenine Cytosine Cytosine Guanine Guanine Thymine Thymine DNA Chromosomes are mainly DNA A single strand of DNA is millions of bases long T ACG Pairing of T on one strand with A on the other, or C with G, causes the double helix to form

31 DNA: a blueprint for making proteins DNA is made up of the 4 bases: A, C, G, and T The order, or “sequence”, of As, Cs, Gs, and Ts on a strand of DNA is really the “instructions” for making proteins GENE A GENE is a piece of DNA that has the instructions for making one specific protein Proteins, like DNA, are long chains of chemicals While DNA is a chain of bases, proteins are chains of AMINO ACIDS AMINO ACIDS There are 20 different amino acids

32 From DNA to protein… Since DNA has only 4 bases, but there are 20 different amino acids, how does the DNA code work?  A group of 3 bases in the DNA sequence is called a “codon” that specifies one specific amino acid  There are also “start” and “stop” signals in the code  “Messenger RNA” or mRNA serves as an intermediate in between DNA and protein

33 mRNA DNA mRNA mRNA has a single strand It faithfully copies the base sequence of one of the DNA strands T In RNA, the base Uracil is used instead of Thymine

34 From mRNA to protein… mRNA Codons Amino acids Folded protein molecule The ALD protein has 745 amino acids! How many bases are needed to encode this protein?

35 The ALD gene Known to be near the tip of the long arm of the Known to be near the tip of the long arm of the X-chromosome X-chromosome “Discovered” in 1993 by Drs. Aubourg, Mandel “Discovered” in 1993 by Drs. Aubourg, Mandel and coworkers: and coworkers:  Location pinpointed more accurately  Sequence of As, Cs, Gs, and Ts (bases) determined determined  The “instructions” for making the ALD protein require 2238* bases! protein require 2238* bases! *For math geeks who know that 745x3=2235 and not 2238, we have to add 3 more bases for the stop signal

36 The ALD gene All ALD patients have a “mutation” in All ALD patients have a “mutation” in their ALD gene: their ALD gene:  An incorrect nucleotide (for example, a “T” where an “A” should be) where an “A” should be)  One or more nucleotides missing  One or more extra nucleotides

37 The ALD protein When there is a mutation in the ALD gene, what happens to the ALD protein? When there is a mutation in the ALD gene, what happens to the ALD protein? Several possibilities: Several possibilities:  A single incorrect amino acid gets placed in the protein (may or may not affect function)  The amino acids are correct up to a point, but then after that they are all incorrect (definitely affects function)  The amino acids are correct up to a point, but then we run into a premature “stop” signal (definitely affects function)

38 The ALD protein contains 745 amino acids The ALD protein contains 745 amino acids Its amino acid sequence was determined from the sequence of bases in the ALD gene Its amino acid sequence was determined from the sequence of bases in the ALD gene ALD protein amino acid sequence was compared to that of other known proteins ALD protein amino acid sequence was compared to that of other known proteins The protein defective in ALD was not any of the enzymes known to participate in the breakdown or synthesis of VLCFA The protein defective in ALD was not any of the enzymes known to participate in the breakdown or synthesis of VLCFA The ALD protein belongs to a large family of proteins called “ABC transporters” The ALD protein belongs to a large family of proteins called “ABC transporters” The ALD protein

39 ABC transporters: Getting something from one side of a membrane… membrane transporter

40 …to another membrane transporter

41 Where is the ALD protein found inside cells? The ALD protein is located in the membrane surrounding “peroxisomes” What are peroxisomes?

42 Cells and their internal structures Smaller compartments within cells are called “organelles” Some of these are: Nucleus Peroxisomes Mitochondria Lysosomes Endoplasmic reticulum Cell membrane

43 Cells and their internal structures Biochemical processes take place within specific organelles Degradation (breakdown) of VLCFA occurs in peroxisomes Synthesis of VLCFA via elongation of LCFA occurs in the endoplasmic reticulum Often, the internal location of a biochemical process is important for understanding how disease results

44 Recent work from the labs of Ronald Wanders and Stefan Kemp in Amsterdam show that the ALD protein transports the “activated” form of VLCFA (VLCFA-CoA) into peroxisomes, where VLCFA breakdown occurs. The ALD protein: What does it do? VLCFA-CoA cytoplasm peroxisome

45 Putting it all together – Normal cells Breakdown of excess ALD Protein VLCFA synthesis (elongation)

46 VLCFA accumulate and are used as building blocks to make complex lipids ALD Protein X Putting it all together – ALD cells VLCFA synthesis (elongation)

47 Another brief summary… Chromosomes are structures in the nucleus of cells that are made up of thousands of genes A gene is a piece of DNA containing “instructions” for making one specific protein If the gene has a mistake in its DNA, the protein that it codes for will often be defective The ALD gene encodes the ALD Protein (ALDP) ALDP is now thought to transport VLCFA-CoA into peroxisomes where degradation takes place

48 Part 3 Prevention and Treatment

49 Prevention and Treatment Knowledge of the biochemistry of ALD has resulted in advances in the prevention and treatment of the disease

50 Prevention Carrier Detection Carrier Detection Prenatal Diagnosis Prenatal Diagnosis  Amniocentesis  Chorion Villus Sampling (CVS) Extended family screening Extended family screening Newborn screening Newborn screening

51 Prevention - Newborn screening “Blood spots” on thick paper cards are obtained from all newborn infants “Blood spots” on thick paper cards are obtained from all newborn infants All states screen for phenylketonuria (PKU) and other treatable genetic diseases All states screen for phenylketonuria (PKU) and other treatable genetic diseases Recent results from the Moser lab demonstrated that screening for ALD is feasible Recent results from the Moser lab demonstrated that screening for ALD is feasible ?Coming soon to a state near you? ?Coming soon to a state near you?

52 Treatment – Diet (Lorenzo’s oil) “Saturated” fatty acid (no double bonds): -C-C-C-C-C-C-C- Saturated VLCFA are toxic to cells “Mono-unsaturated” fatty acid (one double bond): -C-C-C-C=C-C-C-C- Monounsaturated VLCFA are far less toxic “Poly-unsaturated” fatty acid (more than one double bond: -C-C-C=C-C-C=C-C-C Also less toxic Some definitions:

53  Lorenzo’s oil is a mixture of triglycerides containing monounsaturated fatty acids  Because monounsaturated fatty acids are there in excess, the body preferentially uses them to synthesize less-toxic monounsaturated VLCFA (rather than toxic saturated VLCFA) by the elongation process shown in slide #22  Patients taking Lorenzo’s oil have reduced plasma levels of saturated VLCFA, but high levels of mono- unsaturated VLCFA Treatment – Diet (Lorenzo’s oil)

54 Bone Marrow Transplantation Rationale for BMT: Rationale for BMT:  Give patient someone else’s cells which contain good enzyme Procedure: Procedure:  Find donor  Destroy patient’s bone marrow cells  Give patient bone marrow from donor

55 Bone Marrow Transplantation Potential problems: Potential problems:  Finding a donor  Risky – Mortality  Rejection  Graft vs. host disease In which patients is BMT more likely to work?  Fairly strict selection criteria

56 Treatment – Gene Therapy Hope for the present and future Hope for the present and future Problems: Problems:  Get gene to correct cells and keep it there  Once there, get it to work properly  Make sure there are no harmful side effects Remember, too much of a good thing can be just as bad as not having enough… Remember, too much of a good thing can be just as bad as not having enough…

57 The final summary… Knowing something about the biochemistry of ALD allows us to perform tests that are useful in the prevention of ALD Knowing something about ALD biochemistry has also facilitated development of therapies such as Lorenzo’s oil, bone marrow transplantation, and gene therapy Knowing something about the biochemistry of ALD also allows us to search for drugs that may, in the future, be beneficial for treatment

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