Having a child reach toddler age involves many exciting new questions and avenues for parents: What should my child eat? What books should I read to my child? What activities should I start involving him or her in? How should I socially interact my child?
What isn’t exciting is worrying about the following: Placement of a feeding tube? What combination of antidepressants and seizure medications should my child take? If my child stops breathing, do I take him or her to the hospital?
The latter group of questions are asked by a parents who have a child with iNCL (Infantile Neuronal Ceroid Lipofuscinosis), a genetically inherited neurodegenerative disorder. A child is born with the disease and seems to develop normally until about 12 months. The genetic defects begin to physically manifest with regression from walking to crawling, muscle spasms leading to seizures, and gradual blindness by 2 years of age.
The pathogenesis begins with one or more mutations in the CLN1 gene, which encodes a protein called Palmitoyl Protein Thioesterase 1 (PPT1). The gene serves as a blueprint for PPT1 synthesis, which performs a very specific function based on its structure. If a mutation is in the wrong place, it can cripple the protein and deem it useless.
Every protein manufactured in the body has a specific function and location. PPT1 specifically functions in a part of the cell called the lysosome. Lysosomes serve as garbage disposals for the cell, digesting unused and malformed molecular waste. PPT1 is essentially a blade of the garbage disposal, cutting specific parts of proteins so that they can undergo further digestion.
When PPT1 is malformed it does not perform this specific cut on many different proteins, causing them to accumulate into what is seen microscopically as lipofuscin, hence the name lipofuscinosis. The accumulation mostly occurs in the brains of infants with iNCL, which eventually leads to apoptosis (cell death) of brain cells. Progressive research is narrowing the specific locations of this accumulation and its deleterious effects.
Different ares of the brain respond differently to the buildup of lipofuscin and, in turn, display different symptoms. Researchers are trying to pinpoint what brain defects result in muscle spasms, blindness, and brain deterioration.
With new and developing technology, the battle against iNCL is constantly changing. It seems as if there are two types of research being conducted: either to understand the pathogenesis of the disease or to discover new therapeutics to treat the disease.
One cannot be conducted without the other, and researchers are always collaborating to understand the disease more fully. However, this disease encompasses details on the molecular level. Every piece of the puzzle requires endless scrutiny in progression towards a cure. In the past couple of years, many striking discoveries have unveiled mechanisms of the disease as well as possible future treatments.
Exploring the Cause
A problem can’t be resolved without knowing the exact cause. Researchers are narrowing down exactly where a mutated PPT1 protein causes the most problems in different types of non-human animal models.
The Research Group of Glycotechnology and Glycobiology in Japan has published a journal from Ryosuke Midorikawa et al. They have discovered a pathway of retinal degeneration in fruit fly subtypes in an article entitled “Autophagy-Dependent Rhodopsin Degredation Prevents Retinal Degeneration in Drosophila” in 2010.
Whereas the mammalian retinal system differs significantly from that of the fruit fly, the fruit fly retinal system contains many proteins/enzymes that are analogous to humans.
Midorikawa and his research group tested the importance of rhodopsin degradation, an essential process for visual development. The brain must associate different types of visual stimulation by altering its network of neurons in order to process a complete image. The direction a neuron grows, and its connection to other cells, depends on retinal stimulation and the placement and removal of rhodopsin molecules.
iNCL patients have problems within this process; improper degradation confuses the pathway from the eye to the visual cortex. The processing department of images in the brain (the visual cortex) may be working along with eye’s response to light; however, the circuitry is jumbled and can’t transfer the correct information. PPT1, the mutated protein in iNCL, may have an important role in this process.
Midorikawa and his colleagues explored two different routes of rhodopsin degradation, the lysosomal pathway and a backup plan, autophagy. The lysosomal pathway is the normal process to degrading unused proteins within a cellular system. Autophagy is a backup mechanism where the cell digests potentially useful components in order to recycle them for more prominent functions that require immediate attention. Continuous autophagy will result in neurodegenerative defects, and has been associated with PPT1 malfunction.
Midorikawa’s experiment focuses on Phosphatidylserine decarboxylase (Psd), an enzyme which plays an important role in autophagy. Fruit fly mutants lacking Psd showed decreased amounts of rhodopsin degradation through the autophagy process. Psd has genetic interaction with PPT1 in a developing retinal system. A system lacking PPT1 will have problems with autophagy in retinal development which influences the onset of blindness in iNCL victims. These experimental results may provide a pathway to consider when inducing treatments to restore PPT1 function in iNCL patients.
Graduating towards a mammalian system, a journal article written by Shannon L. Macauley at the Washington University School of Medicine in Saint Louis, tests the effects of iNCL patient brain degeneration in her article “Cerebellar Pathology and Motor Deficits in the PPT1-Deficient Mouse.”
Macauley and colleagues explore a region of the brain called the cerebellum. With any networking system, there are a number of different support systems that moderate conductance throughout. In the brain, an important part of the network is a cell type called purkinje, which are moderated by astrocytes and microglia. The purkinje cells transfer information from proliferative cells of the brain to conducting cells in the cerebellum. The cerebellum is an important area for processing coordinated movement and shows degeneration and malfunction in iNCL patients.
Human iNCL patients with defective PPT1 protein show microglial and astrocyte activation. Microglia induce inflammation in the brain, an important process involved in cleaning and repairing problems. Astrocytes nourish neurons and facilitate communication. Both of these cells play beneficiary roles in normal brains, but can cause deleterious effects when over expressed.
Macauley and her team of experts compared these effects with motor coordination in mice of different ages. Results show that there is noticeable purkinje cell loss with 3 month old PPT1 deficient mice in comparison to mice with normal levels of PPT1. The purkinje cell loss correlates with poor motor coordination of mice during experimentation. At 7 months of age, PPT1 deficient mice had cerebellums that weighed half the amount of normal mice, on average.
This proves a vital piece of information, as many therapies have specific targets in specific locations. Macauley and colleagues enhance the ability to explore specific regions of the brain in specific time periods, an integral piece to therapy.
Another novel journal article in 2010 shows that polyunsaturated fatty acids have therapeutic effects on cultured cells of PPT1 individuals. Sung-Jo Kim and fellow scientists at the National Institute of Health and Child Development explain test results in their article “Omega-3 and Omega-6 Fatty Acids Suppress ER- and Oxidative- Stress in Cultured Neurons and Neuronal Progenitor Cells from Mice Lacking PPT1.”
When cells are in problematic conditions, they become stressed, just like people. Molecular interactions don’t match up perfectly and a cascade of problems can occur from a single issue. When neurons become stressed they produce substances called reactive oxygen species. Reactive oxygen species are molecules that have an extra electron in their molecular orbit. This extraneous electron can grab onto a number of different corresponding hosts, damaging the host and its relationship.
Polyunsaturated Fatty Acids (PUFAs) are a natural product made by the body to defend itself from reactive oxygen species. PUFAs are also found in high concentrations of developing neuronal systems including gene expression.
Kim first showed that there are increased levels of reactive oxygen species in PPT1 deficient cells and explains, “this result suggests that.. cortical neurons are under oxidative stress.”
Kim injected PUFAs into a growing cell lacking PPT1. Each cell developed with fewer amounts of reactive oxygen species and resistance to apoptosis (cell death). Addition of PUFAs also increased levels of heat shock protein 32, a type of protein that provides additional defense against reactive oxygen species.
So studies in 2010 have provided significant insights into developmental problems and a hint to how they might be solved.
Now comes the big problem: how to cross the blood brain barrier.
The blood brain barrier is constructed to highly regulate to what can access the brain. Most chemicals cannot cross this barrier effectively, which poses a major problem for therapeutics. Thankfully a father son team, the Dawsons, are working on this problem.
A solution to the problem: The Dawsons
Glyn Dawson, PhD at the University of Chicago, has been studying various forms of NCL for over 15 years.
His son, Phillip Dawson PhD, runs a laboratory at Scripps Medical Institute in San Diego, CA. Their collaboration in 2010 resulted in a journal entitled “PPT1 Inhibitors Can Act as Pharmacological Chaperones in Infantile Batten Disease.” Phillip Dawson was willing to clarify results and discuss potential therapy at his lab in November, 2011.
The article discusses the concept of chaperones, which are mild inhibitors of the target protein. What would debilitate a normal protein by its chemical interaction, chaperones actually increase functionality in mutated proteins. This is analogous to the effect of a crutch. Use of a crutch will slow down a healthy person, but increases mobility in a handicapped person. Therefore, the chaperone only works on proteins with partial functionality.
P. Dawson explains, “there is a mutation which manufactures the protein halfway and then stops, we can’t do anything about that. But, there are a significant number of missense mutations where the protein is just a little bit destabilized [and] is prematurely degraded.. Hopefully, we can tickle that pathway a little bit.”
The Dawsons did indeed tickle that pathway. Their test results show that use of chaperones restored PPT1 function two-fold. “It has been shown that you don’t need much enzyme activity to make it to adulthood,” P. Dawson says, “A significant change can be from 3% to 6%.”
The solution has been proven in cultured cells outside of the body. Now, the issue is finding a safe method to integrate it past the blood brain barrier into the brain. Intracerebral injection is one method (injection directly into the brain), but it poses infectious risk. It is also not attractive to pharmaceutical companies, which are the driving factors taking a therapy from research to clinical trials.
Kathy Partin , a PhD at Colorado State University with successful neurological drugs in the latter stages of clinical trials explains, “for a drug company to invest in making a drug there can’t be too much risk... it’s true that doctors can deliver a drug directly to cerebrospinal fluid, it’s also true that it’s expensive and risky.”
Dawson also address that issue with the use of quantum dots.
“Quantum dots are small particles of metal surrounded by organic material that can attach different substrates,” Dawson explains, “It is then a matter of finding something of the same physiological characteristic that is biodegradable.”
Finding that substitute is more easily said than done. After finding a proper substitution, it must be then tested in all avenues to ensure that it is effective and safe.
Then comes the problem of funding from pharmaceutical companies. All of these techniques seem amazing, revolutionary, and contain much potential, but one has to tease potential into reality in order to receive funding. Even then, the patients are children and tests can’t proceed based simply on potential.
Partin clarifies the situation, “We, as a society, have decided that drug development is under capitalistic rules. We don’t want the government to subsidize it. The problem... is that they make business decisions based on business outcomes and not individual outcomes.”
In all hope, the potential does exist. It is just a matter of supporting it. Fortunately, the exploration of technology with this disease which is so specific, is paving the way for treatments in other neurological diseases such as Huntington’s Disease and Alzheimer’s Disease, which helps popularize such a specific and rare disease.
Interest of the public transforms into interest of researchers and pharmaceutical companies. WIth the proper support and encouragement from a large group of informed individuals, it can happen sooner than later. Patients with iNCL, who rarely age into double digits, have a limited amount of time.
• Daniel Gutman, a Mesa native, Mountain View High School alum, and friend to the Taylor family, studied Batten Disease while completing his master’s degree in biomedical sciences from Colorado State University last year.