Molecular and Cellular Mechanisms of Learning Disabilities: A Focus on Neurofibromatosis Type I

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Molecular and Cellular Mechanisms of Learning Disabilities: A Focus on Neurofibromatosis Type I Carrie Shilyansky1, Weidong Li1, M. Acosta2, Y. Elgersma3, F. Hannan4, M. Hardt5, K. Hunter-Schaedle6, L.C. Krab3, E. Legius7, B. Wiltgen1 and Alcino J Silva1 1

Department of Neurobiology, University of California, Los Angeles Department of Neurology, Children’s National Medical Center, Washington DC 3 Neuroscience Institute, Erasmus University, Rotterdam 4 Cell Biology and Anatomy Department, New York Medical College, New York 5 Department of Psychology and Clinical Neuroscience Laboratory, University of California, Los Angeles 6 Children’s Tumor Foundation, New Jersey 7 Department of Human Genetics, Catholic University of Leuven, Leuven 2

INTRODUCTION Introduction to learning disabilities NF1 AND LEARNING DISABILITIES NF1 AND ADHD OTHER NF1 PHENOTYPES MODEL STUDIES OF NF1: DROSOPHILA AND MICE ANATOMICAL CORRELATES OF LEARNING DISABILITIES AND COGNITIVE SYMPTOMS IN NF1 MOLECULAR MECHANISMS UNDERLYING THE COGNITIVE DEFICITS IN NF1 REVERSING THE MOLECULAR, PHYSIOLOGICAL AND COGNITIVE DEFICITS OF NF1 WITH STATINS MEASURING EFFICACY IN STATIN CLINICAL TRIALS THE ROLE OF PATIENT ORGANIZATIONS IN STUDIES OF LEARNING DISABILITIES FINDINGS FROM STUDIES OF NF1: IMPLICATION TO LEARNING DISABILITIES GENETIC AND PHARMACOLOGICAL MANIPULATIONS THAT ENHANCE LEARNING AND MEMORY: AN ALTERNATIVE STRATEGY REFERENCES

Animal and Translational Models for CNS Drug Discovery, Vol. 2 of 3: Neurological Disorders Robert McArthur and Franco Borsini (eds), Academic Press, 2008

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INTRODUCTION Learning disabilities (LD) affect approximately 15% of the US population, but currently there are no generally effective therapies for this class of cognitive disorders. To address this problem, we have initially focused on Neurofibromatosis type I (NF1), a single gene disorder associated with learning disabilities. Previous studies showed that mice with a heterozygous null mutation of the NF1 gene have behavioral deficits related to the cognitive phenotype of NF1 patients. Studies of these mice showed increased Ras/MAPK signaling in the hippocampus and prefrontal cortex which results in the upregulation of GABA release underlying the spatial, attention and working memory deficits of the mutants. Statins can be used to decrease the isoprenylation and therefore the activity of Ras. Recent results demonstrate that a short treatment with statins, which is ineffective in controls, can reverse the increases in Ras-MAPK signaling, the synaptic plasticity, attention, and spatial learning deficits of the Nf1 mutant mice. To test whether statins can also reverse the neurological and cognitive deficits associated with NF1 in patients, we have initiated a series of collaborative pilot clinical studies in children and adults with NF1. These studies will not only have an impact on our understanding of cognitive deficits associated with NF1, it will also serve as a case study for how to investigate and treat learning disabilities.

Introduction to learning disabilities Learning disability is a class of neurological disorders affecting approximately 15% of the US population. The diagnosis of learning disability is broad; it reflects difficulties in learning of specific academic skills in subjects with otherwise normal cognition. Learning disabilities differ from mental retardation or learning problems caused primarily by visual, auditory, or motor handicaps. Unlike these other disorders, learning disabilities are characterized by specific impairments restricted to certain domains of mental function. Therefore, individuals with learning disabilities have multiple areas of strengths, but their academic performance is confounded by significant weaknesses in certain skill sets. The simplest definition of a learning disorder is a discrepancy between tests of intellectual capability and actual achievement.1 Intellectual capability is typically quantified with IQ tests like the Wechsler scales (WISC-IV, WAIS-III) or the Stanford Binet (SB-IV). These tests provide reliable measures that are standardized across specific age groups.2 Academic achievement is also determined using standardized tests that assess performance level in multiple academic areas (e.g., Woodcock-Johnson (WJ-III)2 or WIAT.3 Learning disability is diagnosed when performance in tests of achievement is significantly below predictions based on IQ score. Beyond this general definition of learning disability, several systems attempt to categorize the disorder into subtypes. The DSM-IV places learning disabilities into three major categories: reading disorder, mathematical disorder, and disorders of written expression.1,4 These categories are determined by the specific set of cognitive abilities that are impaired in an individual. In addition to these categories, the DSM-IV also recognizes non-verbal learning disorders and learning disorder not otherwise specified. These latter categories represent learning disabilities which cause a pattern of weaknesses

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that do not fall neatly into one of the first three DSM-IV categories of learning disability.5 The DSM-IV categories are also considered distinct from other developmental disorders such as motor skills disorders and communication disorders. Other diagnostic systems are based on specific descriptions of the skills that are most impaired by the learning disability; For example, dyslexia (reading disorder), dysgraphia (writing disorder), dyscalculia (problems with arithmetic and math), dyspraxia (motor coordination).4 Attention deficit disorder often occurs in individuals with learning disorder, but is considered a separate, co-morbid diagnosis. In addition, the etiology of learning disabilities is multivariate, making the problem even more complex. In some genetic or neurological conditions, specific learning problems are part of the phenomenology associated with the diagnosis. However, in other diagnosis a non-specific impairment defines the associated learning disability. In practice, definitions regarding impairments in learning are complex and require a careful evaluation of the cognitive profile of the affected individual, including the performance area that is impacted (e.g., difficulties in visuospatial organization could result in deficits in mathematical abilities and in phonological decoding required for reading). A careful assessment can provide a better overview of the profile of strengths and difficulties and allow the clinician to understand a changing pattern of individual performance in response to increasing demands from the environment. Careful description of the impairments of individuals with learning disability is important, because it allows appropriate intervention and support to be implemented. Parents and school teachers would be able to make accommodations according to the weaknesses of an individual. Such an individualized program allows learning to be less dependent on weaker cognitive abilities, and the individual can use their strengths to bypass potential deficits. These support-based interventions improve the achievement of an individual with learning disabilities. However, they require a lifelong, intensive commitment from an individual with learning disability as well as their parents and teachers. Further, the effectiveness of the intervention depends on the accuracy and completeness with which the learning disability subtype is diagnosed and strengths and weaknesses of the student are described. The diagnostic criteria described above identify patterns of learning problems, but may not capture the entire clinical picture. It is unclear whether diagnostic criteria reflect fundamental differences in underlying neuronal mechanisms. A better understanding of the neuronal mechanisms that are impaired in learning disability disorders could lead to more accurate diagnosis and the development of more effective treatments.

NF1 AND LEARNING DISABILITIES The causes for learning disabilities are multivariate. However, several neurological diseases of known etiology have learning disability as a prominent symptom. NF1 is one such disease. NF1 is diagnosed by the presence of a combination of symptoms in multiple organ systems. As a model of learning disabilities, NF1 provides an important advantage as it is caused by mutations to a single known gene. Importantly, it is associated with a very high frequency of learning disabilities (30–80%) and a low frequency of mental retardation (6–8% compared with 4% in the general population).

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This wide range of estimated learning disability is due in part to studies using differing definitions of a learning disorder, as well as use of small groups without appropriate controls.6,7 It is important to note that patients with NF1 do not show pronounced deficits on IQ tests and other neuropsychological measures sensitive to overall cognitive impairment.8 Although specific, learning disabilities and cognitive impairments in patients with NF1 do limit their academic and social gains, sometimes quite severely. In NF1 patients, impairments greater than predicted based on IQ are seen in four main cognitive areas: visuospatial function, executive function and planning, attention, and reading/vocabulary.9 NF1 has been historically associated with deficits in visuospatial skills. Several studies have found consistent deficits in the Judgment of Line Orientation (JLO) test in this patient population. However, JLO requires more than just visuospatial skills. Recent studies have demonstrated that the pattern of learning and cognitive deficits in patients with NF1 is more complex than those detected by JLO, and that it includes visuospatial organization, motor abilities, and social skills. Most notably, NF1 is associated with robust deficits in tests of visuospatial functioning and impaired performance in spatial learning tests.7,8 Also, patients with NF1 demonstrate deficits in organizational skills and planning. Further, they have notable social problems, related to impairments in attention and perception of social cues.6,7 In addition, NF1 patients demonstrate language-based learning problems. Patients with NF1 have deficits in expressive and receptive language, vocabulary, visual naming, and phonologic awareness. In fact, reading and spelling are repeatedly found to be impaired more severely than predicted by IQ in NF1.9 Consistent with these impairments in language-based learning, NF1 patients show poorer academic achievement in reading and writing, compared to their unaffected siblings. However, in NF1 patients, there appears to be no discrepancy between verbal IQ and performance IQ.6 Hence, performance-related impairments and motor coordination problems are also common in NF1. The pattern of learning disabilities seen in NF1, as it is the case with other disorders, does not fall cleanly into one of the DSM-IV defined categories. Instead, the occurrence of multiple types of learning problems suggests that NF1 impairs a fundamental learning process.

NF1 AND ADHD In addition to learning disabilities, deficits in other behavioral and cognitive domains can limit academic and social achievement in patients with NF1.This has been highlighted by studies showing that up to 40% of children with NF1, who were identified as academic underachievers, were nevertheless normal on neuropsychological tests for learning disabilities.8 In such cases, underachievement may be caused by deficits in attention, planning, and organization skills,7–9 which are consistently seen in NF1 patients. There is a high co-morbidity between NF1 and attention deficit disorder. Studies have reported incidence rates of 30–50% of attention deficits in NF1 populations,9–11 an occurrence three times more frequent than that observed in unaffected siblings.9 Attention deficit in NF1 has been found at early ages and persists throughout adulthood. The inattentive subtype of ADHD is most frequently present in NF1 patients,

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followed by the combined subtype.9 The effects of attention deficit in children with NF1 are quite broad, and they may affect both academic and social learning. Children with NF1 tend to have social problems and appear socially awkward and withdrawn, in comparison to their siblings and to children with other chronic, life-threatening illnesses.12 Parents commonly complain that their children with NF1 are unable to engage in successful peer relationships.8 Some evidence suggests that social deficits are related to poor interpersonal skills, which may occur as a result of decreased attention to social cues. When social skills are assessed using the Social Skills Rating System, children with ADHD and NF1 have the poorest outcomes compared to children with NF1 alone or with NF1 and learning disabilities.12,13 Among the many symptoms associated with NF1, co-morbid attention deficit disorder in NF1 is increasingly recognized as having dramatic and important effects on quality of life for affected individuals.i

OTHER NF1 PHENOTYPES Aside from the cognitive and behavioral phenotypes, NF1 is associated with a myriad of symptoms affecting multiple organ systems, including tumors, bone changes, and cardiovascular problems. The NF1 gene codes for a tumor suppressor and therefore, loss of the wild-type NF1 allele in a variety of cell types results in tumor formation.14 Benign nervous system tumors such as neurofibromas and gliomas are frequent in NF1. Some other tumors associated with NF1 are pheochromocytomas, gastrointestinal stromal tumors, non-ossifying fibromas of bone, and glomus tumors of the finger tips. Malignancies seen at increased frequency in NF1 include peripheral nerve sheath tumors, glioblastoma multiforme, rhabdomyosarcoma, and juvenile myelomonocytic leukemia.15 Abnormalities of melanocyte proliferation are virtually always present and result in café-au-lait spots, skin fold freckling, and iris hamartomas (Lisch nodules).16 The skeletal phenotype in NF1 is characterized by mild short stature, osteopenia, and pectus excavatum. In some patients a local bone dysplasia can result in severe scoliosis, sphenoid bone dysplasia, or pseudarthrosis.17NF1 mutations also affect the cardiovascular and cerebrovascular system resulting in an increased frequency of congenital pulmonic valve stenosis, essential hypertension, renal artery stenosis, and cerebrovascular malformations.18,19 Several growth, cardiovascular, cognitive, cutaneous, and facial characteristics of NF1 are similar to other conditions resulting from genetic abnormalities in the RAS-MAPKinase pathway such as LEOPARD syndrome (PTPN11), Noonan syndrome (PTPN11, KRAS), Cardio-facio-cutaneous syndrome (BRAF, KRAS), and Costello syndrome (HRAS). These conditions have been grouped under the name of “neuro-cardio-facial-cutaneous” (NCFC) syndromes.20 Neurofibromin, the protein product of the NF1 gene, is a negative regulator of an important signal transduction pathway (RAS-MAP kinase pathway). The central role of this pathway in cellular function is reflected in the many different clinical problems

i For further discussion of learning disabilities, please refer to Tannock et al., Perspectives on ADHD: An integrative assessment of the use of animal models for developing novel therapeutic agents and Bartz et al., Preclinical animal models of autistic spectrum disorders (ASD) in Volume 1, Psychiatric Disorders.

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that can be seen in individuals with NF1. Animal models of NF1 have provided extensive insight into the mechanisms by which changes in this pathway result in the clinical manifestations of NF1.

MODEL STUDIES OF NF1: DROSOPHILA AND MICE Animal models of NF1-related learning disorders offer many advantages, including rapid testing of learning and memory behaviors in genetically well-defined organisms, with rigorous experimental controls. In addition to its effects on Ras, NF1 also controls adenylyl cyclase (AC) activity in both fruit flies and mammals.21–23 A novel growth factor stimulated AC pathway that requires both NF1 and Ras has been recently identified in flies.23 There are also at least two separate neurotransmitter activated G-protein dependent AC pathways in flies that are controlled by NF1.23 NF1 acting via Gsα and the Rutabaga AC is required for olfactory learning in flies,24 while the NF1/Ras pathway is necessary for long-term memory in flies.25 Strikingly, separate domains of the NF1 protein control each pathway since the GAP domain alone is sufficient for longterm memory, while the C-terminal portion is essential for learning.25 The fruit fly long-term memory task is equivalent to the mouse spatial learning task, since both require multiple rounds of training and are dependent on protein synthesis.26,27 Both the Ras and the AC pathways are important for hippocampal-dependent spatial learning in mice.28 Further, studies of spatial learning defects in Nf1 heterozygous mice (Nf1⫾) show that pharmacologic or genetic manipulation of the Ras pathway can rescue learning in these mice.29–32 Currently available therapeutics such as lovastatin, a cholesterol lowering agent, decrease Ras activity, and fully rescue the spatial learning defect in Nf1⫾ mice.32 The current statin clinical trials for learning disabilities associated with NF1 were directly stimulated by these most recent findings in mice.32 Thus, animal models of NF1 have had a central role in therapeutic development for NF1.

ANATOMICAL CORRELATES OF LEARNING DISABILITIES AND COGNITIVE SYMPTOMS IN NF1 Converging evidence from both human and animal studies implicates the hippocampal, prefrontal, and cerebellar systems in the learning disabilities and cognitive problems associated with NF1. Learning and remembering new facts and general information about the world requires the hippocampus and surrounding medial temporal cortex.33,34 Spatial learning tasks are known to depend on the hippocampus.35,36 Damage to the hippocampus produces profound amnesia for spatial information and difficulties navigating through space. Also, patients with damage to this area often have considerable difficulty learning and retrieving new vocabulary words.37 Although considerably milder than patients with lesions, the pattern of deficits in NF1 suggests that hippocampal-dependent domains are affected, such as spatial processing as well as reading comprehension and vocabulary learning in school. Learning disabilities associated with NF1, therefore, may be better understood by considering how NF1 affects hippocampus function, physiology, and ability to process information.

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Data from human studies suggests that the prefrontal cortex also plays a critical role in many of the cognitive processes affected in NF1. Symptoms seen in NF1, such as increased impulsiveness, attention deficit, and difficulties with planning and behavioral organization, are commonly seen in syndromes involving prefrontal dysfunction. Finally, NF1 patients display symptoms suggestive of cerebellar dysfunction. NF1 is associated with a variety of fine and gross motor function impairments, including problems with fine motor coordination, hypotonia and problems with balance and gait.38–41 Half of the NF1 children seen at the Sophia’s children hospital/Erasmus MC is scored “clumsy” on physical examination by a pediatric neurologist and almost 60% of these patients require physiotherapy.42 There are several candidate brain regions that could play a role in the motor deficits in NF1, such as the premotor- and association areas, the primary motor cortex, the basal ganglia, and different sub-regions of the cerebellum.43 The cerebellum is of special interest in NF1, since this region is critically involved in fine motor control. Although NF1 patients are not clearly ataxic, NF1-related hypotonia and clumsiness can very well arise from dysfunction of the vermis and/or intermediate zone of the cerebellum.43 Moreover, the cerebellum is one of the predominant sites for NF1-related hyperintensities visible on T2-weighted MRI, which have been implicated in fine-motor problems.44 To understand how NF1 affects hippocampal, prefrontal, and cerebellar circuits it is critical to understand how this protein modulates molecular and physiological function in these structures.

MOLECULAR MECHANISMS UNDERLYING THE COGNITIVE DEFICITS IN NF1 Studies in the mouse model of NF1 have revealed how changes in the NF1/ras pathway cause behavioral learning deficits associated with the disease. The NF1 mouse model (Nf1 ⫾ mouse) shows behavioral phenotypes in several cognitive domains affected by NF1 in patients. These phenotypes include impairments in spatial learning,29 attention,32 and possibly behavioral planning. Spatial learning and memory in mice requires experience-dependent plasticity in the hippocampus. During learning, synapses in the hippocampus undergo long-term potentiation (LTP), a persistent, activitydependent increase in synaptic strength. Nf1⫾ mice have impaired hippocampal LTP, which very likely causes their spatial learning impairments in behavioral tasks. LTP deficits in the Nf1⫾ mice are caused by an abnormally high level of activity of the RAS/MAP Kinase pathway. The Nf1 gene product, neurofibromin, is a negative regulator of the ras signaling pathway.45,46 Therefore, loss of neurofibromin function in NF1 leads to increased ras signaling.32 LTP deficits can be rescued in the Nf1⫾ mice by manipulations which normalize the level of ras activity. These same manipulations also rescue behavioral impairments in spatial learning.32,47 Behavioral data from the Nf1⫾ mouse model also suggests that ras-dependent prefrontal dysfunction contributes to impairments in attention and planning. Nf1⫾ mice show impairments in behavioral tests of attention and planning which depend on prefrontal cortex. For example, the NF1 mouse shows impairments in the lateralized reaction time task, a test of visuospatial attention32 developed from a task that is sensitive to lesions and manipulations of prefrontal function in humans.48–50 Performance

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deficits of the Nf1⫾ mice in this task are also ras dependent, similarly to the spatial learning impairments,32 since these deficits can be rescued by manipulations that decrease ras activity. Therefore, the Nf1⫾ mouse model demonstrates that both learning and attention deficits in NF1 are caused by increased ras activity. Increased ras levels in the Nf1⫾ mouse cause an abnormal increase in inhibitory activity in the hippocampus.47 This high level of inhibition disrupts activity-dependent plasticity in the hippocampus, causing the spatial learning impairments in the Nf1⫾ mice. Ras-dependent increases in activity of inhibitory networks may also occur in other brain areas where increased RAS activity is seen in NF1. This includes the prefrontal cortex, where increased RAS activity was shown in the Nf1⫾ mouse, and where balanced inhibition is thought to be critical for function. This may also be expected to include the cerebellum. Purkinje cells, which are the sole output of the cerebellar cortex, are GABA-ergic neurons, and among the highest neurofibromin expressing cells in the brain.51,52 In prefrontal cortex and cerebellum, ras-dependent increases in inhibition offer a compelling potential mechanism by which NF1 deletion can disrupt the physiological function of these structures to cause multiple cognitive symptoms of NF1 such as attention, planning, and motor coordination impairments.

REVERSING THE MOLECULAR, PHYSIOLOGICAL AND COGNITIVE DEFICITS OF NF1 WITH STATINS As stated above, the key pathophysiologic mechanism underlying multiple NF1 symptoms in both humans53,54 and mice47,55–57 is Nf1-dependent Ras/MAPK activity. Data from the mouse model of NF1 clearly place increased ras activity as central to cognitive symptoms of NF1 such as attention and learning deficits.32,47 Therefore, therapeutic interventions designed to inhibit p21Ras function have been proposed as treatments for NF1.58 Post-translational isoprenylation is required for the membrane localization and function of p21Ras, and isoprenylation provides a potential target for NF1 pharmacotherapy.59 Indeed, pharmacologic inhibitors of farnesyltransferase downregulate p21Ras activity and can rescue physiological and spatial learning deficits associated with Nf1⫾ mice.47 It is unknown, however, whether any of these inhibitors have the in vivo pharmacokinetics, biodistribution, and safety profile required for the long-term treatment of cognitive dysfunction in NF1.60 Fortunately, other pharmacologic agents with better-established safety profiles, such as the statins, are also able to regulate p21Ras isoprenylation. Isoprenyl groups are synthesized during an intermediate step in cholesterol synthesis. Statins, cholesterol lowering agents widely prescribed to treat hyperlipidemia,61 inhibit cholesterol synthesis by irreversibly blocking the rate-limiting enzyme in this process (HMG-CoA reductase). In this manner, statins are able to decrease the availability of isoprenyl groups and thus lower p21Ras isoprenylation.62,63 Lovastatin, a statin which passes the blood–brain barrier, was shown to inhibit the enhanced p21Ras-MAPK activity in the cortex and hippocampus of Nf1 ⫾ mice32 at doses that do not seem to affect controls. In preclinical experiments using the mouse model of NF1, lovastatin was found to effectively reverse molecular, physiological, and behavioral impairments. Even a short 4-day treatment with lovastatin in adult

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Nf1 ⫾ mice normalized Ras activity and LTP at hippocampal synapses. Lovastatin treatment also rescued behavioral impairments, reversing both spatial learning and attention deficits.32 These results, taken together with the fact that lovastatin is a widely prescribed drug that is known to be well-tolerated even in long-term treatments, make lovastatin a viable potential treatment option for cognitive deficits associated with NF1 in humans. These preclinical findings have spurred multi-center clinical trials examining the safety and efficacy of lovastatin treatment for cognitive symptoms in the NF1 patient population. This represents the first scientifically based pharmacological treatment for learning disabilities. Positive results could foment tests of this and perhaps other pharmacologic agents in other learning disabilities. As with NF1, the evolving richness of the genetics and pharmacology of memory could be used to identify the molecular, cellular, systems, and cognitive mechanisms underlying learning disabilities. This would mark a paradigm shift for helping the 6% of children in public schools currently receiving help for their learning disabilities (about 2.8 million in 2002 in the USA alone).64

MEASURING EFFICACY IN STATIN CLINICAL TRIALS In clinical trials designed to assess the effect of statins on cognition, it is of pivotal importance to use a carefully designed battery of tasks. These tasks should be sensitive, quantitative and have high test–retest reliability. These tasks should also address robust, core features and underlying mechanisms of the cognitive symptoms seen in patients. Also, it is important to include tests that closely reflect the cognitive domains that have already been shown to be responsive to statins in mice. Clinical trials designed in this way could provide sensitive and clearly interpretable results. In this design, disease mechanisms described in the NF1 mouse model guide the choice of both the therapeutic being tested (lovastatin) and the clinical endpoints being used to measure drug efficacy. Two experimental tests that can contribute to the available test battery are working memory and motor learning tests, both of which are developed to assess specific impairments that are found in NF1 patients and studied in animal models. Prefrontal cortex dysfunction is thought to contribute to many of the cognitive symptoms seen in NF1 patients. Importantly, preclinical studies showed that lovastatin rescued prefrontal RAS/MAPK dysfunction, as well as deficits in attention in Nf1 ⫾ mice.32 Therefore, clinical trials could utilize tasks, such as working memory tasks, which are known to require prefrontal function and have been shown to be sensitive to prefrontal impairment in other disease states.65,66 Performance in specific working memory tasks reflects prefrontal cortex engagement and function.67 A battery of such working memory tasks has been designed to isolate two distinct facets of working memory, maintenance and manipulation. Working memory maintenance can be tested by asking subjects to mentally maintain the spatial location of several items (1–7) in memory over a short time. Manipulation of information in working memory can be tested by asking subjects to mentally flip the spatial items over a horizontal line. Such tasks should be paired with an equivalent verbal task to explore whether any observed deficit is specific to spatial encoding. By changing the number of items

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presented, these tasks allow working memory load to be parametrically varied, ensuring that even minor behavioral deficit can be detected.65–67 This battery of tasks isolates specific processes of working memory, while retaining the sensitivity to reveal small changes in working memory ability associated with drug treatment. In a clinical trial, the sensitivity of these measures is critical given that the drug dose being tested may not have been optimized and so treatment-related improvements may be relatively small. Such working memory tests can be included in a clinical trial as one measure of the efficacy of lovastatin in improving prefrontalrelated cognitive symptoms in NF1. Since motor skills are impaired in NF1 patients, adding a parameter of motor functioning is important when assessing the effect of statins in NF1. In addition to tests for fine motor skills, such as the Beery Visual Motor Integration test, functional integrity, and plasticity in the brain areas involved in motor skills can be assessed by testing adaptation of motor skills (i.e., motor learning). One motor learning test that could be applied to this purpose is the prism adaptation test, a quantitative, easy and quick way to measure the adaptation of eye–hand movements to visual displacement of the environment by prism glasses. Prism adaptation is thought to be dependent not only on plasticity in the anterior and caudal posterior lobe of the cerebellar cortex, but also on plasticity in various other motor areas.68,69 Since prism adaptation can be rapidly learned, well-quantified and is not sensitive to a placebo or test–retest effect it is ideal measures to include in the assessment of drugs that potentially improve brain function.

THE ROLE OF PATIENT ORGANIZATIONS IN STUDIES OF LEARNING DISABILITIES The child with NF1 is at risk for physically devastating and potentially life-threatening neurological tumors, bone deformities, and an array of other complications. It is unpredictable if and when these might appear, and there are still no effective NF1 drugs. For many parents, however, one of the first (and for many, the most distressful) issues they confront after NF1 diagnosis is being told their child may “struggle in school”. Again unpredictable, this may range from mild to severe. Fortunately, there are organizations such as the Children’s Tumor Foundation and NF Inc. that offer support networks and information, such as the location of NF clinics and the medical resources patients need. These networks of support are a very important and effective way to organize and empower patients, and to provide the support they need in dealing with the significant life-changing problems associated with NF1. Patient organizations are also having an increasingly active role in all aspects of research and development. They are an important source of motivation and inspiration for scientists working in the area. They often provide much needed pilot resources that allow laboratories to venture into a particular area of research, and they facilitate the process of translating basic laboratory findings into clinical trials. Patient organizations have also had important roles in lobbying governmental organizations and obtaining the required support for research. In this respect, these organizations can also have a critical role in facilitating the complex process of drug development and implementation of new treatments.

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It is critical that patient organizations are active participants in the complex, tortuous and often lengthy process of developing treatments for disorders as complex as learning disabilities. NF1 is a wonderful example of how the involvement and commitment of families of affected individuals can have an enormous impact on research and development.

FINDINGS FROM STUDIES OF NF1: IMPLICATION TO LEARNING DISABILITIES Most learning disabilities have no known etiology or treatment and they can impact every aspect of an individual’s life. The emerging biology of learning and memory – which spans molecular, cellular, systems, and behavioral findings – promises to revolutionize the study and treatment of learning disabilities. These studies have uncovered a number of molecular, cellular, and systems mechanisms that could account for the cognitive profiles observed in learning-disabled patients. Specific ion channels, neurotransmitters, receptor complexes, signal transduction mechanisms, transcription factors, and genes, have become part of the fast evolving molecular puzzle of memory.70 Furthermore, environmental influences add complexity to this tantalizing puzzle. It is very likely that genes primarily responsible for one learning disability may also contribute to natural variation of the phenotype in another learning disability. Additionally, genes that affect any one aspect of a learning disability may also have an impact on other phenotypes associated with the disability, or could even contribute or exacerbate other neurological or psychiatric disorders. There is a considerable amount of insight into the molecular mechanisms disrupted by NF1 mutations and these have been instrumental in unraveling both the cause of the disorder and in developing treatments. Furthermore, studies of the NF1 learning and memory deficits have revealed the involvement of mechanisms previously uncovered in more general studies of learning and memory. Thus, the emerging biology of learning and memory is useful in understanding the underlying causes of learning disabilities, and the study of these disorders also promises to be broadly relevant to other learning disabilities and to our understanding of normal learning and memory.

GENETIC AND PHARMACOLOGICAL MANIPULATIONS THAT ENHANCE LEARNING AND MEMORY: AN ALTERNATIVE STRATEGY Current understanding of the neurobiology of learning can also be used to devise strategies for cognitive enhancement which would not require identification of underlying disease causing mechanisms. This approach is important since the majority of learning-disabled individuals are not part of clearly identified groups, and their disorders do not have known etiologies. Such general enhancement strategies may be valuable not only for learning disabilities but also in other disorders associated with cognitive dysfunction, particularly those of heterogeneous or undefined etiology. For example, genetic analysis has shown that schizophrenia71 and depression72 are heterogeneous conditions, each caused by the cumulative effects of numerous mutations

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and many incompletely understood developmental and environmental factors. In such cases, as in many learning disability subcategories, it is unclear which single mechanism or molecular pathway should be targeted for pharmacotherapy. Thus, in developing therapeutics for such disorders, it is worthwhile to consider strategies to improve cognitive function which could be used irrespective of detailed knowledge of the underlying pathophysiology. It is important to note that the FDA has already approved a number of drugs that are thought to enhance cognitive function, including donepezil (Aricept®), rivastigmine tartrate (Exelon®), galantamine HBr (Razadyne®), memantine (Namenda®), and modafinil (Provigil®).73 These and other compounds could be tested for their usefulness in treating learning disabilities. Molecular and cellular studies into cognitive enhancements may identify additional targets for therapeutics aimed at potentiating learning processes to compensate for the disruptive effects of disease causing mutations. Enhancing the strength of induction of learning processes may compensate for downstream deficits. Alternatively, enhancing the sensitivity of the final molecular events in synaptic plasticity and learning may be able to compensate for disease-related impairments in induction. Currently, there is considerable evidence for animal models with dramatic cognitive enhancements.28 In several of these animal models, genetic and pharmacological manipulations that enhance induction of LTP also facilitate behavioral learning and memory.28 The over-expression of genes known to be required for LTP induction can result in enhancements in LTP and learning. For example, activation and opening of the NMDA receptor (NMDAR) is a critical initial step in the induction of LTP. The transgenic over-expression of the NMDAR subunit 2B enhances the opening time of the NMDAR. Such transgenic mice show enhancements in LTP and several forms of behavioral learning.74 Further, the mutation of negative regulators of LTP induction can also lead to learning enhancements. The nociceptin receptor normally acts as a negative regulator of LTP induction. Mice carrying inactivating mutations of nociceptin show increased hippocampal LTP as well as improved spatial learning and memory.75 Learning enhancements are also seen in mice with genetic manipulations of downstream effectors of LTP. Tissue plasminogen activator (TPA) is an extracellular protease that is thought to be involved in synaptic remodeling triggered by plasticity and learning. Such synaptic remodeling is thought to maintain learning in a neuronal network. Transgenic over-expression of TPA enhances LTP expression and behavioral learning.76 In contrast, a null mutation of this gene impairs these two phenomena.77 Thus, molecules such as TPA may modulate the downstream processes involved in expression and maintenance of synaptic plasticity. Such molecules may represent a potential molecular target which can enhance the ability of even impaired learning processes to become effectively encoded. The list of known memory-enhancing mutations is by no means limited to the examples presented. Other genetic manipulations which enhanced LTP and learning targeted pre-synaptic Growth Associated Protein 43,78 adenylyl cyclase,79 telencephalonspecific cell adhesion molecule,80 calcineurin, Rim 1 (a ras effector), and forebrain H-rasG12V. 81 Finding common molecular, cellular, and systems changes underlying the learning and memory enhancements in this diverse collection of mutants may be especially informative for developing future approaches to identifying novel therapeutics for learning disabilities.

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