Neurofibromatosis (NF) is a set of three genetic diseases affecting over 1 in 3,500 people in the United States alone. These diseases are characterized by the growth of benign tumors in the peripheral nervous system. NF is often visible as skin abnormal­ities and sometimes associated with early cognitive and sensory disabilities. The precise symptoms, affected regions and molecular mechanisms of disease pathology vary by type. This review will focus on dysfunctions caused by loss-of-function mutations in each form of NF within Schwann cells. In NF-1, the first-discovered and most common type of NF, a mutated NF1 gene truncates the neurofibromin protein. The reduction or loss of NF-1 expression in cells increases RAS pathway activity, leading to excess prolifer­ation and differentiation. In NF-2, the mutated NF2 gene results in dysfunctional Merlin protein, which normally regulates cell adhe­sion and contact inhibition by inhibiting kinase activation. NF-3, the rarest and most recently discovered type, is due to SMARCB1 (INI1) tumor suppressor gene loss and LZTR1. LZTR1 genes encode for schwannomatosis. However, an underlying mechanism for NF-3 is still unknown. Currently, treatments are nonspecific for benign tumors, including surgery and tumor-suppressing drugs. With future research into mechanisms behind each type of NF, researchers may develop more effective and specific therapies.

Introduction

Neurofibromatosis (NF) is a set of three genetic diseases af­fecting over 1 in 4,000 people in the United States alone. There are three different variants that fall under the same neurofibromatosis category with the most well-known being NF-1. NF-1 is the most common of the three and affects about 1 in 3,000 individuals. NF-2 was later discov­ered and has also been studied in depth. About 1 in 25,000 individuals suffer with this form of neurofibromatosis. The last variant, NF-3, is not as well- studied. The symptoms of NF usually appear by the age of 10 and a child is 50% more likely to inherit this disease if a parent has one mutated gene. The symptoms of this disease are usually characterized by the growth of benign tumors in the peripheral nervous system (Ahn MS, et al., 1996). About 5-10 percent of these NF tumors become malignant. Schwannomatosis is also a characteristic that occurs in about 15% of all neurofibromatosis cases because schwann cells are greatly affected in NF. Schwannomatosis is a very serious condition that consists of multiple cutaneous “schwannomas” which are dangerous to the central nervous system. NF is often visible as skin abnormalities and may be associat­ed with early cognitive and sensory disabilities. The precise symptoms, affected regions and molecular mechanisms of disease pathology vary by type. NF diseases are usually diagnosed according to standardized clinical criteria or genetic testing. Genetic testing is very useful and may detect about 95% of mutations but cannot test for the severeness of the disease. (Spyk et al., 2011).

The genetics of NF vary. However, all of them consist of loss-of-function mutations in tumor-suppressor genes. In NF-1, there is a loss of function in the neurofibromin protein. This alters the Ras pathway, leading to excess proliferation. In NF-2 there is a loss of function in the merlin protein.This impaired merlin protein cannot inhibit Rac, impairing contact inhibition and ultimately leading to excess proliferation. Genetic mecha­nisms are not as well known for NF-3. It is known that loss of function of the SMARCB1 protein leads to tumorigenesis. Although researchers are beginning to understand more about the molecular mechanisms of the disease, treatments for all three NF diseases are widely unspecific. The current most common treatments consist of tumor-specific drugs with an­giogenesis inhibitors. Other treatments include cancer approaches such as radiation, chemotherapy and surgery to remove tumors (Lin, 2013) (Asthangiri et al., 2009).

Figure 1A. Normal schwann cell-developing neurofibromas. Through loss-of-function mutations that occur in NF1, NF2, and NF3 genes, normal Schwann cells develop neurofibromas which lead to tumorigenesis.

Figure 1B. Protein dysfunction in the three types of NF leading to Schwann cell tumorigenesis. In NF-1, impaired NF1 protein increases activity in the Ras pathway, leading to excess proliferation. In NF-2, impaired Merlin protein cannot inhibit Rac, impairing contact inhibition. In NF-3, it is known that an impaired SMARCB1 protein leads to tumorigenesis, but the precise mechanism is not yet fully known.

 

Figure 2. Ras activity in the MAPK/ERK signalling pathway leads to proliferation. Activation of Ras by receptor-tyrosine kinase (RTK) causes an interaction with Raf ki­nase which phosphorylates a mitogen-activated protein kinase (MEK). MEK then phosphorylates an extracellular signal-regulated kinase (ERK). ERK then enters the nucleus and phosphorylates transcription factors, such as CREB, Myc, and Elk-1, which promote synthesis of proteins that are involved in cell proliferation. Neurofibromin 1 (NF1) acts to prevent excess proliferation by inactivating Ras.

 

The MAPK/ERK Signal Pathway

Dysfunctions in the MAPK/ERK signaling pathway have been implicated in many forms of cancer due to upregulated Ras activity. This pathway is important for regulating cell survival, proliferation and differentiation by manipulating protein synthesis. In healthy cells, an extracellular mitogen binds to a receptor on the membrane surface. This causes the GTPase Ras to exchange its bound GDP for GTP so that it may activate Raf kinase and begin an intracellular signalling cascade. Raf kinase phosphorylates mitogen-activated protein kinase (MEK), which in turn phosphorylates extracellular signal-regulated kinase (ERK). ERK can then phosphorylate a multitude of transcription factors such as Myc, CREB, and Fos to regulate the transcription of proteins that lead to cell proliferation (Hood et al., 2003). Unchecked proliferation is the first step in the process of cancer development due to mutations in tumor-suppressor genes, causing a loss-of-function. In the MAPK/ERK pathway, this loss-of-function causes Ras to retain its bound GTP, continuing this unregulat­ed cell proliferation.

NF-1

NF-1 or neurofibromatosis 1 is the first disease within the NF triad. NF-1 alone affects about 1 in 3,000 people. About 50% of these cases are inherited and 50% occur spontaneously. Symptoms usually appear by age 10 and vary in severity. Symptoms may run from flat, light brown spots on the skin to freckles in the armpits or groin area. Soft bumps under the skin called neurofibromas are also likely to occur. Some symptoms, however, may not be identified as quickly. These range from bone deformities (such as growth deficiency in bone minerals) to tumor growth in the optic nerve. Learning disabilities are unfortunately another symptom of this disease (Ahn MS, et al., 1996). According to the Mayo Clinic, a diagnosis of ADHD in these individuals is very common.

NF-1 is diagnosed according to standardized clinical criteria. These criteria were established in 1987 by the National Institute of Health. According to these criteria, NF-1 may be diagnosed if any two features of the above symptoms are present. NF-1 is generally characterized by the brown spots and freckles on the skin along with appearance of neurofi­bromas. Being diagnosed with NF-1 is not an immediate death sentence but it may shorten the life of an individual by 15 years (Spyk et al., 2011). Although genetic testing may detect about 95% of the mutations associat­ed with NF-1, it does not predict the severity of NF-1. However, if doctors notice a deletion of genetic material that includes the NF-1 gene and many surrounding genes (this is the cause in about 3-5% of individuals), these people will generally have a much more severe phenotype.

On the subject of the genetics of NF-1, it is known that muta­tions of the NF-1 gene (which encodes for the neurofibromin protein) lead to neurofibromatosis in individuals (Bollag, et, al., 1996). Neurofibromin contains 2,818 amino acids and is expressed at low levels in healthy individuals. It functions as a negative regulator for the cRas/MAPK sig­naling pathway. Neurofibromin contains a Ras-specific GTPase activating protein domain which interacts with Ras. This can be seen in both sup­plemental figures. This interaction increases GTPase activity of the Ras protein, ultimately increasing the conversion of active GTP-bound Ras to inactive GTP-bound Ras. This change from active to inactive leads to a decrease in cell signaling. The Ras signaling pathway is extremely critical for controlling cellular growth, proliferation and differentiation. This lack-of-function neurofibromin results in more activation of this signaling pathway, leading to excess proliferation and cell growth and ultimately causing the development of tumors (Spyk et al., 2011).

NF-2

After the identification of neurofibromatosis, it was discovered that another form of the disease exists known as neurofibromatosis type 2 (NF-2). This type is rarer than NF-1, occurring in approximately 1 out of 25,000 people, and it can become visible later than NF-1 with signs sometimes appearing in patients up to 55 years old (Evans et al., 2005). NF-2 can be distinguished from other forms of neurofibromatosis by somewhat different pathology. Like NF-1, schwannomas are common in NF-2, with other glial tumors such as astrocytomas forming more rarely. However, in NF-2, it is significantly more common for tumors to form in or around the cranial nerves, especially the vestibulocochlear nerve, rather than in the peripheral nervous system. As a result of the dysfunction in some of these nerves, sensory impairment is a common symptom of NF-2, including cataracts, progressive hearing loss and retinal tumors (Asthangiri et al., 2009). On the other hand, cognitive impairments are not seen as commonly in NF-2.

The root causes of NF-2 are missense mutations in the NF2 gene (Yang et al., 2011). These mutations can be found in NF-2 patients and occasionally in cases of other tumor types, including mesotheliomas (Bianchi et al., 1995). The NF2 gene encodes for moesin-, ezrin-, and radixin-like protein, also known as merlin, which loses proper function in NF-2 (Trofatter et al., 1993; Rouleau et al., 1993). The importance of merlin dysfunction to the disease’s pathology has been demonstrated in a study where the protein’s activation is artificially blocked, resulting in tum­origenesis. When the proper function of merlin was restored, the tumors were suppressed (Jin et al., 2006).

Merlin’s tumor-suppressor properties are related to contact inhi­bition of proliferation and are most likely connected to its role in regulating cell-cell adhesion. This function is highlighted in a study showing that merlin is highly expressed in cases of tissue fusion (McLaughlin et al., 2007). Since the protein’s discovery, several different molecular mecha­nisms have been found to contribute to merlin’s tumor suppression.

One study performed on cultured human kidney cells showed that merlin can interact with mixed-lineage kinase 3 (MLK3) to inhibit the kinase’s normal function of promoting activation by Raf (Chadee et al., 2006). As shown in Figure 2, Raf is an essential component in the signaling cascade of the MAPK/ERK pathway that can lead to prolifera­tion. By interacting with MLK3, merlin inhibits one step in this pathway, making it more difficult for tumors to form. Other research has found that merlin may also suppress tumors by inhibiting a different step in the same pathway. By inhibiting Rac in NF2 deficient cells, it was demonstrated that the tumorigenic effect cannot occur without Rac, as it is responsible for activating Ras. This indicates that merlin interacts with Rac in some way to prevent Ras’s activation, as shown in Figure 1B (Bosco et al., 2010). In the case of NF-2, the dysfunctional merlin is unable to properly interact with the MAPK/ERK pathway through either of these mechanisms to suppress tumor formation.

NF-3

The rarest and most recently discovered form of neurofibroma­tosis is schwannomatosis. Affecting around 1 in 40,000 individuals (Me­lean, Sestini, Ammannati, & Papi, 2004), this form of neurofibromatosis has been the recent subject of study in order to determine its molecular mechanism. Schwannomatosis is characterized by the presence of benign tumors known as schwannomas located in the peripheral nervous system, similar to neurofibromatosis type 2 (NF-2). The areas common­ly affected include the spine and peripheral nerves, with cranial nerve schwannomas being less common (Kehrer-Sawatzk, Farschtsch, Maut­ner, & Cooper, 2017). Anatomically, the tumors either localize to a single limb or spread throughout the spine or half the body. (Kehrer-Sawatzk, Farschtsch, Mautner, & Cooper, 2017).

Due to overlapping clinical features, schwannomatosis was originally undifferentiated from NF-2. However, about two-thirds of NF-2 patients have tumors located subcutaneously rather than near the cranial nerves (Jacoby et al., 1997). This finding ignited a study conducted using patients who met the researcher’s criteria for schwannomatosis. In order to determine the relationship between schwannomatosis and NF-2, the NF2 locus was examined for mutations similar to those in NF-2. Analysis of the NF2 locus revealed that similar truncating mutations seen in NF-2 were occurring in schwannomatosis patients with varying patterns of in­activity that significantly differed from NF-2 (Jacoby et al., 1997). Another study that attempted to differentiate NF-2 and schwannomatosis exam­ined the clinical phenotypes of families in the United Kingdom (U.K.). Six families with seven sporadic cases of the disease were studied. It was found that hereditary mutations in schwannomatosis were only slightly similar to NF-2 (Evans et al., 1997). Along with this genetic difference, a clinical difference in terminology was used to differentiate between the tumors in NF-2 and schwannomatosis because the classification of neurofibromas in schwannomatosis was found to be incorrect. The proper classification involved schwannomas, highlighting another difference between the two disease forms (Evans, et al., 1997).

The molecular and genetic basis of schwannomatosis was unknown until the link between schwannomatosis and SMARCB1 was discovered. SMARCB1 is implicated in the development of malignant rhabdoid tumors in the brains and kidneys of children and is almost always fatal (Smith et al., 2012). In a study, mRNA was isolated from blood samples of four Italian families harboring schwannomatosis and meningiomas. These samples ran through RT-PCR for sequencing of SMARCB1 and NF2. A point mutation in exon 1 of the SMARCB1 gene, wherein a glutamine switches to a valine at the 31st position, was found in the affected members of the studied families (Bacci et al., 2010). The same mutation was observed by studying the schwannomas extracted from other affected family members (Bacci et al., 2010). The nine coding exons of SMARCB1 were studied in 56 French patients who met the clin­ical definition for schwannomatosis. Nine sequence variations were found between all patients, including variations that led to a truncated protein that lost its function (Rosseau, Noguchi, Bourdon, Sobol, & Olschwang, 2011). These mutations were found in both sporadic and familial forms of the disease, including patients whose onset occurred at the age of 35. (Rousseau et al., 2011). Interestingly, no NF2 germline mutations were found in schwannomatosis, unlike the germline SMARCB1 mutations described above (Smith et al., 2012).

Along with SMARCB1, two other genes have been implicated in schwannomatosis: COQ6 and LZTR1. COQ6 is an important enzyme involved in the synthesis of coenzyme Q10, an enzyme heavily implicated in the electron transport chain and mitochondrial function. Point mutations in this protein have been discovered in families with schwannomatosis (Zhang et al., 2014). Additionally, heterozygous loss-of-function muta­tions for LZTR1 have been discovered in schwannomatosis patients, providing further evidence that an interaction of multiple genes is involved in the complex mechanism of schwannomatosis (Smith et al., 2015). Therefore, the proposed mechanism of schwannomatosis is the inter­action of multiple genes in addition to common NF2 mutations found in NF-2; SMARCB1, LZTR1, and COQ6 mutations play a role in the devel­opment of tumors in a way that is different than rhabdoid tumor formation (Kehrer-Sawatzk, Farschtsch, Mautner, & Cooper, 2017).

Treatment

Today, treatments for all three types of neurofibromatosis are still largely nonspecific to the disease. The most common pharmaceutical treatments are nonspecific tumor-suppressor drugs, with the angiogene­sis inhibitor bevacizumab being the most frequently prescribed. Patients who suffer from NF-induced tumors that are severe enough to interfere with their life commonly have the tumors surgically removed, which can often prevent sensory impairments but carries its own risks.

In the future, more effective therapies may specifically target dysfunctional pathways in NF pathology. Drugs may be developed to either replace the mutated protein for each type of the disease, replicate their specific tumor suppressive function, or inhibit the MAPK/ERK path­way to inhibit the tumorigenesis found in neurofibromatosis (Lin, 2013; Asthangiri et al., 2009).

Conclusion

In conclusion, neurofibromatosis is a set of three conditions (NF-1, NF-2, and schwannomatosis) that cause tumor growth within the nervous system. Originally categorized as NF-1 and NF-2, schwannoma­tosis was differentiated from NF-2 by different tumor locations. A common pathology of each condition includes the development of tumors and chronic pain. As with many cancers, the molecular mechanism proposed for neurofibromatosis is the dysfunction of the MAPK/ERK signaling pathway that causes unchecked proliferation in Schwann cells. This dysfunction has been linked to mutations that cause tumor-suppressor proteins such as NF, merlin, SMARCB1, and LZTR1 to lose their function, causing widespread growth. Currently, targeted treatments do not exist for neurofibromatosis, but in the future, such therapies may target specific tumor-suppressors in order to restore their function.

Acknowledgements

Our group would like to thank Dr. Shubhik DebBurman for his insight, guidance, and encouragement. We would also like to thank our peer teacher, Sierra Smith ’17, for her feedback. Finally, we would like to thank the Lake Forest College Biology Department and Neuroscience Program for this opportunity to expand our knowledge on brain diseases.

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