April is Parkinson's Awareness Month

  • An Estimated 10 Million People Worldwide Have Parkinson’s Disease (PD)
  • A Cure for PD Has Yet to Be Discovered
  • Short-Interfering RNA (siRNA) Knockdown and mRNA-Based Cell Reprogramming Are Shown to Be Promising Approaches 

Every April, the Parkinson’s Foundation engages the global Parkinson’s community to support Parkinson’s Awareness Month in an effort to raise awareness about Parkinson’s disease (PD) and how the Foundation helps make lives better for people with PD. The stated aim is to “do more together to improve care and advance research toward a cure.” This year the theme is “Start a Conversation,” a call to action that urges people to talk about Parkinson’s with their loved ones, friends, neighbors, care team and community. In keeping with this theme, the present blog provides a brief overview of PD followed by two nucleic acid-based therapeutic approaches: short-interfering RNA (siRNA) and mRNA-induced cell differentiation.

 

Brief Overview of PD

Statistics: The following information about PD was selected from a lengthy summary provided by the U.S. National Institute of Neurological Disorders and Stroke (NINDS), except where indicated. PD is the second-most common neurodegenerative disorder in the U.S., following Alzheimer’s disease. Most people diagnosed with PD are age 60 years or older; however, 5-10% of people with PD are diagnosed before the age of 50. Experts estimate that as many as 1 million Americans have PD, and an estimated 10 million people worldwide are living with PD. The number of people diagnosed with PD in the U.S. is expected to double by 2040. The combined direct and indirect cost of PD is estimated to be nearly $25 billion per year in the U.S. alone. 

Symptoms: By the time a diagnosis is made, people with PD typically exhibit difficulty controlling the movement of their bodies due to tremors (involuntary shaking), bradykinesia (slowness of movement and reflexes), and impaired balance. As these symptoms progress, walking, talking, swallowing, and completing other simple tasks can become challenging.

Pathology: The nervous system is made up of individual units called nerve cells or neurons. Neurons serve as a "communication network" within the brain and throughout a person’s body. The symptoms of PD result primarily from the death of neurons in the substantia nigra, a region in the midbrain critical for motor control. Neurons use chemical messengers called neurotransmitters to send information between neurons by crossing the space between them, called the synapse. Normally, neurons in the substantia nigra produce a neurotransmitter known as dopamine (DA).

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DA is critical for movement and it helps transmit messages within the brain to make sure muscles produce smooth, purposeful movement. Loss of DA results in abnormal nerve firing patterns that impair movement. By the time Parkinson’s is diagnosed, most people have lost an estimated 60 to 80 percent of their DA-producing cells in the substantia nigra.

A factor believed to play a fundamental role in the development of PD involves abnormalities of a 140-amico acid protein called alpha-synuclein (aSyn). In the normal brain, aSyn is located in nerve cells in specialized structures called presynaptic terminals. These terminals release neurotransmitters which then carry signals between neurons. This signaling system is vital for normal brain function.

Evidence suggests that the buildup of misfolded aSyn plays a key role in the development of PD. As the misfolded protein accumulates, it clumps together into aggregates that join together to form tiny protein threads called fibrils. Fibrils are the building blocks for Lewy bodies (LB), abnormal structures that form inside nerve cells in the substantia nigra and elsewhere in the brain. LB are a pathological hallmark of PD. In this regard, I found a very informative 2016 PhD thesis by Eleonora Carboni on X-ray visualization of aSyn aggregation stating that “whether the aggregated forms of aSyn present in LB are per se toxic, is still under debate, but there is a large consensus that aggregated forms of aSyn, like oligomers, are able to exert cellular toxicity in invitro and in vivo.”

Genetic Studies: In most instances the cause of PD is unknown, although a small proportion of cases can be attributed to genetic factors. An estimated 15-25% of people with PD have a family history of the disorder. It is relatively rare for PD to be caused by a single mutation of one of several specific genes. This only accounts for about 30% of cases in which there is a family history of PD and only 3-5% of sporadic cases, i.e. instances with no known family history.

Researchers increasingly believe that most, if not all, cases of PD probably involve both a genetic and environmental component. Based on an analysis of genome-wide association data (GWAS) for people with PD, scientists have identified >41 loci believed to be independently associated with PD risk. The NeuroChip microarray recently reported by Blauwendraat et al. enables accurate identification of over 5.3 million common single nucleotide polymorphisms (aka SNPs). According to Blauwendraat et al., this comprehensive content makes the NeuroChip “a reliable, high-throughput, cost-effective screening tool for genetic research and molecular diagnostics in neurodegenerative diseases” including PD. 

Mitochondrial Dysfunction: Research suggests that damage to mitochondria plays a major role in the development of PD. Mitochondria are unique parts of the cell that have their own DNA  that is entirely separate from the genes found in the nucleus of every cell.

Mitochondrial dysfunction is a leading source of free radicals—molecules that damage membranes, proteins, DNA, and other parts of the cell. Oxidative stress is the main cause of damage by free radicals. A number of  genes found to cause PD disturb the process by which damaged mitochondria are disposed of in the neuron (mitophagy).

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According to an article in Genetic & Engineering News, the results of a recent study have   provided some insight into the mechanisms by which a particular form of aSyn (aSyn*) exerts its toxic effects that include the phosphorylation of several kinases of the MAPK pathway, as well as the formation of another Tau protein form at the mitochondrial membrane, likely contributing to mitotoxicity. 

Cell-to-cell Transmission of Abnormally-folded Proteins: Researchers have learned more about how PD-related damage spreads to various parts of the brain and nervous system. Recent evidence suggests that even before LB are detectable in the brain, aSyn aggregates and LB can be found in the nervous system of the gastrointestinal (GI) tract and in the salivary glands, a finding that supports the theory that PD many originate not in the brain but in the autonomic nervous system. Non-motor symptoms such as constipation may in fact be a sign of the disease affecting nerves outside the brain before the disease moves into the brain where it later affects regions that control movement. Researchers hypothesize that abnormal aSyn may act like a “seed” causes other aSyn molecules to misfold, leading to a cell-to-cell transmission of PD-like brain changes, especially in regions of the brain important for motor function.

Drugs for PD

A comprehensive review of the current and candidate drugs for PD was published in 2017 by Hannah Sally. In a nutshell, 35 drugs are presently available for PD patients; however, a cure for this disease is yet to be discovered. Patients depend on symptom ameliorating drugs, and PD therefore represents a large unmet medical need. As of 2017, 224 drugs were in active development for PD, with 68% of all drugs being in preclinical stages. The 11 drugs in late stage development (Phase III and pre-registration) have one thing in common with each other, and current drugs on the market: they all act upon the dopaminergic system in the brain. Following are brief overviews of two nucleic acid-based approaches to treat PD using siRNA and mRNA-induced cell differentiation. 

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siRNA Targeting aSyn

Nucleic acid-based approaches to drugs for PD have been reviewed by Jamebozorgi et al. in 2018 in Molecular Neurobiology, and by Nakamori et al. in 2019 in Neurotherapeutics. Of these strategies, reduction of expression of aSyn represents a promising therapeutic strategy. Specific reduction of gene expression can be achieved by using synthetic non-coding siRNAs against the target mRNA, thereby taking advantage of triggering endogenous RNA interference (RNAi). However, efficient delivery of siRNA into neurons in vivo remains challenging due to biological barriers, degradation, low transfection efficacy, and insufficient distribution.

The polyethylenimine (PEI) complexation of siRNA into PEI/siRNA nanoparticles provides a non-viral nucleic acid delivery platform. PEIs are positively charged polymers that form non-covalent complexes with nucleic acids, thus protecting siRNAs from degradation, mediating cellular uptake, and efficiently promoting lysosomal protection and escape into the cytoplasm.

It has been previously established that the 4–12 kDa branched PEI F25-LMW, a low-molecular weight PEI with superior transfection efficacy and low toxicity, is useful for delivery of siRNA in vivo and in vitro. Helmschrodt et al. have now reported that siRNA complexed with this PEI extensively distributes across the CNS down to the lumbar spinal cord (show here) after a single intracerebroventricular infusion.

pastedGraphic_4.png MRI scan of lumbar spine.

In brief, siRNA against aSyn was complexed with PEI F25-LMW and injected into the lateral ventricle of mice overexpressing human wild-type aSyn (Thy1-aSyn mice). Five days after the single injection of 0.75 μg PEI/siRNA, aSyn mRNA expression in the striatum was reduced by 65%, accompanied by reduction of aSyn protein by ∼50%. Mice did not show signs of toxicity or adverse effects. Moreover, ependymocytes and brain parenchyma were completely preserved and free of immune cell invasion, astrogliosis, or microglial activation. Helmschrodt et al. concluded that these results support the efficacy and safety of PEI nanoparticle-mediated delivery of siRNA to the brain for therapeutic intervention. 

mRNA-Induced DA Neurons for PD

According to a 2017 publication by Kim et al., generation of functional DA neurons (shown here) is an essential step for the development of effective cell therapy for PD. The generation of DA neurons can be accomplished by overexpression of DA-inducible genes using virus- or DNA-based gene delivery methods. However, these gene delivery methods often cause chromosomal anomalies. In contrast, mRNA-based gene delivery avoids this problem and thus is considered safer to use in the development of cell-based therapy. Kim et al. therefore used  mRNA-based gene delivery to generate genomic integration-free DA neurons by transfection of mRNA encoding DA-inducible genes Nurr1 and FoxA2.

pastedGraphic_5.png 3D computer illustration of isolated DA neurons on a white background.

Use of these two genes was discussed in earlier published studies by others. Nurr1 is a transcription factor essential for midbrain-specific DA neuron development and can induce transplantable DA neurons from rat neural precursor cells (NPCs). The additional expression of FoxA2 further improved the yield, survival, and function of Nurr1-induced DA neurons. Kim et al. found that the delivery of mRNA encoding these two dopaminergic-fate inducing genes proved sufficient to induce naive rat forebrain precursor cells to differentiate into neurons exhibiting the biochemical, electrophysiological, and functional properties of DA neurons in vitro. 

The generation efficiency of DA neurons was improved by the addition of dibutyryl cAMP (db-cAMP). This cAMP derivative is known to stimulate protein kinase A (PKA) signaling,  which leads to gene expression activation. When db-cAMP was added to mRNA-transfected rat NPCs, Kim et al. observed significant enhancement of protein expression in rat NPCs. In the presence of db-cAMP, the expression of NURR1 and FOXA2 proteins was increased by 2- and 2.3-fold, respectively. In a separate but related study of mRNA transfection by Baek et al., it was demonstrated that production of induced neuronal cells alleviated PD symptoms in a mouse model.

According to Kim et al., their data show that proteins can be stably translated from in vitro synthesized mRNA and can be efficiently expressed in rat NPCs with the addition of db-cAMP. Moreover, “the successful generation of DA neurons using an mRNA-based method offers the possibility of developing clinical-grade cell sources for neuronal cell replacement treatment for PD.” This latter possibility has indeed been realized, as described in the next section.

PD Stem Cell Therapy Begins

A November 14, 2018 news item in Nature magazine reported that Japanese neurosurgeons have implanted ‘reprogrammed’ stem cells into the brain of a patient with PD for the first time. This approach is conceptually similar to the work of Kim et al. but used mRNAs encoded in DNA vectors, and reprogrammed skin cells from an anonymous donor into induced pluripotent stem cells (iPSCs), which can be made to ‘morph’ (i.e. differentiate) into neurons that produce DA for transplant.   

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In October 2018, 2.4 million DA precursor cells were transplanted into the brain of a patient in his 50s. In the three-hour procedure, the surgical team deposited the cells into 12 sites known to be centers of DA activity. Implanted DA precursor cells had been previously reported to improve symptoms of PD in a monkey model (see Footnote below).

According to this news report in Nature, the patient is doing well and there have been no major adverse reactions so far. Researchers will observe the patient for six months and, if no complications arise, will implant another 2.4 million DA precursor cells into the brain of the patient. The team plans to treat six more patients with PD to test the technique’s safety and efficacy by the end of 2020, with potential commercialization by 2023, depending on how good the results are.

Concluding Comments

I hope that this blog has provided you with an increased awareness of PD from a number of medical and technical perspectives. I am optimistic about future therapies for PD using siRNA, mRNA-reprogrammed dopaminergic cells, and possibly a combination of these treatments. However, to me, the complexity and invasive nature of such clinical procedures beg the question of whether systemic delivery to the target region of the brain might be possible using aptamer or antibody conjugates, which I have previously blogged about here.

As usual, your comments are welcomed.

Footnote

The following is the previously reported method for implantation of DA precursor cells in a monkey model of PD: 

“The coordinates of the targets were obtained using MRI, and human iPS cell-derived dopaminergic neuron progenitors were stereotactically transplanted into the putamen of the MPTP-treated monkeys bilaterally.”   

The putatem is labeled in the depiction of the brain provided at the top of this blog.

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