Neurocognitive endophenotypes in CGG KI and Fmr1 KO mouse models of Fragile X-Associated disorders: an analysis of the state of the field

Battery development and implementation

The first step in evaluating the potential for a behavioral phenotype in CGG KI mice was to choose what cognitive domains to test. Unfortunately, at the outset there was a startling lack of data on which to base experimental hypotheses, both from human or mouse research. There were anecdotal data from patients that suggested problems with "foggy thinking" in Premutation carriers, which was interpreted by our clinical collaborators as a combination of abnormal hippocampal and parietal cortex function. A separate collaborator of ours that was interested in neurocognition set out to identify any potential prodromal neurocognitive deficits in Fragile X Premutation carriers, focusing primarily on spatiotemporal processing^49–51. Using both of these pieces of anecdotal evidence in conjunction with the preliminary data from our collaborators as support for our hypothesis, we set out to evaluate the potential for spatial and temporal processing deficits in CGG KI mice. Similarly, we extended our analysis to visuomotor processing as motor learning and motor performance have a strong dependence on intact spatiotemporal integration^40,43.

More to the point, we examined spatial and temporal processing deficits in CGG KI mice using behavioral tasks designed to test specific hypotheses about the cognitive strengths and weaknesses reported in Fragile X Premutation carriers without FXTAS. These cognitive domains were chosen because a number of neurodevelopmental disorders share fundamental spatiotemporal processing deficits described as a spatiotemporal hypergranularity⁵². This hypergranularity, or reduced resolution in temporal and spatial attention, impairs spatiotemporal processing that underlies memory formation. What this means is that a greater difference or separation among elements in space or time is required before they can be discriminated by the research subject.

Spatial processing. Using a pair of behavioral tasks designed specifically to evaluate resolution of spatial processing in CGG KI mice (Figure 1A-B), it was demonstrated that CGG KI mice show deficits in processing the specific distances that separate two objects in space using a metric change detection task (also called a coordinate change). These deficits were present in male CGG KI mice as early as 3 months of age, and persisted until at least 12 months of age^41,44.

Figure 1. Paradigms used to evaluate spatial and temporal processing in mice.

A. Metric or Coordinate change detection task. B. Topological or Categorical change detection task. C. Temporal order for visual objects task. D. Novelty detection for visual objects task. All of these paradigms rely on spontaneous exploration to guide behavior, and thus are not confounded by alterations in affect or the requirement to food deprive animals to provide motivation to perform a task to receive a food or water reward.

Performance was also tested in a topological spatial memory task that required the mice to remember the relative spatial locations of two dissimilar objects after their positions were transposed (also called a categorical change). Male CGG KI mice did not show deficits at 3 or 6 months of age, but did show deficits when they were 9 and 12 months of age compared to age-matched wildtype littermate controls (Figure 2)^41,44.

Figure 2. Spatial processing deficits in CGG KI mice scale with CGG repeat length and age.

A. Metric/Coordinate task by repeat length and age. B. Topological/Categorical task by repeat length and age. The red line signifies no response from the animal to spatial change. Data replotted from Hunsaker et al., 2009, 2012^41,44.

The results from these spatial experiments suggest that: (1) the resolution of spatial processing in CGG KI mice is reduced at a very young age. This suggests the spatial resolution is due to abnormal development, as this reduced spatial resolution appears to be then be maintained over time, meaning it does not progressively worsen with aging, but does show a relationship with CGG repeat length. (2) General spatial memory as measured by the topological change detection task does show progressive worsening across age as well as showing a relationship with CGG repeat length, with deficits emerging in adults and becoming more profound with advancing age in CGG KI mice.

Temporal processing. To evaluate temporal processing in CGG KI mice, a temporal ordering for visual objects task was used. In this task, mice are presented sequentially with three different pairs of identical objects, and time spent exploring the objects is recorded. The mice are then presented with one object from the first pair and one object from the third pair, and time spent exploring is again recorded. Typically, mice preferentially explore the first over the third object. At least 24 hours later to reduce mnemonic interference, after a different set of objects have been presented, the mice are presented with the first object they were presented with that day as well as a never-before-seen novel object. Mice preferentially explore the novel object over the familiar one. On the novelty detection test the CGG KI mice show more exploration of the novel object as do wildtype littermates, but unlike wildtype mice do not show an object preference in the temporal ordering test (i.e., tested with the first and third presented object). This pattern of results indicates that object recognition is intact, but the processing of temporal order is impaired (Figure 3)^42,44.

Figure 3. Temporal processing deficits in CGG KI mice scale with CGG repeat length and sex.

A. Temporal Ordering for Visual Objects Task in male mice. B. Temporal Ordering for Visual Objects Task in female mice. C. Novelty Detection for Visual Objects Task in male mice. D. Novelty Detection for Visual Objects Task in female mice. Data replotted from Hunsaker et al., 2010, 2012^42,44.

An important aspect to these behavioral results is that both male and female CGG KI mice showed deficits. This is important because female Premutation carriers generally show reduced disease severity due to the protective effect of a second, non-mutated FMR1 gene on the inactive X allele (i.e., reduced genetic dosage)⁵⁶. The presence of temporal processing deficits in both male and female CGG KI mice suggests impairments to these processes are fundamental consequences of the Premutation, since deficits are present and identifiable even in the least affected subgroup within the population. Similar effects are currently being identified and characterized in human Premutation carriers both symptomatic and asymptomatic for motor features associated with FXTAS^49–51.

Motor performance. To evaluate potential subclinical gait ataxia or general clumsiness in the CGG KI mice similar to anecdotal reports from our clinical collaborators, a skilled ladder-walking task has been employed. In this test mice are allowed to walk across a series of very thin ladder rungs perpendicular to the direction of travel (similar to walking across a ladder set on the ground). The number of times that the mouse makes an error in foot placement is recorded and is operationally defined as a foot slip. To perform these tasks, the mouse is placed at one end of the apparatus and allowed to cross from one end to the other into a darkened box⁴³.

The apparatus used in these experiments was modified from previously described ladder rung tasks^70–72. The apparatus had black plastic walls separated by approximately 5cm, with 2mm diameter rungs making up the floor. For this initial study, the mice were placed at one end of the apparatus and were allowed to walk back and forth for 2 minutes. The number of foot slips was recorded for the 2 min duration, except when the mouse was turning around. The number of times the mouse traveled from one end of the apparatus to the other was also recorded as a general locomotor measure. On this task, CGG KI mice as young as 2 months of age already showed an increased number of foot slips compared to wildtype litter mate controls (Figure 4)⁴³. Importantly, the CGG KI mice showed more of both forelimb and hindlimb slips than wildtype mice, suggesting both visuospatial and basic motor deficits. That is, forelimb foot slips may suggest visuospatial processing deficits, such as difficulty in planning forepaw placement as well as difficulty updating movements as the step progressed (i.e., as the mouse moved forward the initial planned step has to be modified subtly and an inability to do so results in a foot slip). Hind foot slips however, have less of a visuospatial planning component, but may reflect a subtle motor effect. This is because stepping with the hind limbs has been shown to be more efficient and easier for the mouse to perform cf.⁴³. Difficulty in hind limb stepping may therefore reflect some form of ataxia, and may underlie impairments in rotarod and stationary beam walking reported previously⁷³. Similar to the metric or coordinate task data described above, the lack of an age effect suggests that these motor features are due to abnormal development, not an age-related progressive impairment.

Figure 4. Performance of male and female CGG KI mice on the ladder rung task separated by sex.

There were no effects of age present in the data. Data replotted from Hunsaker et al., 2011⁴³.

Demonstrating a neurobehavioral endophenotype

Behavioral analysis. After the initial development of the neurocognitive endphenotype described above in the CGG KI mouse model of the Fragile X Premutation, it became clear that these experiments were begging replication in the Fmr1 KO mouse model of Fragile X Syndrome. We posited two possible outcomes for such a replication and extension study, the first being that the CGG KI mouse and the Fmr1 KO mouse would show completely independent and non-overlapping deficits wherein CGG repeat dosage would be non-predictive of cognitive deficits. The second suggestion was that the CGG KI mouse and the Fmr1 KO mouse may show a predictable relationship with increasing CGG repeat length in CGG KI mice and Fmr1 KO mice showing a linear or otherwise parametric association with each other.

To test these possibilities, we combined our published CGG KI results⁴⁴ with the data generated by other labs^47,48 wherein they used the battery described above in Figure 1 to evaluate the cognitive deficits in the Fmr1 KO mouse as part of interventional treatment studies. Overall, the data support both of the proposed outcomes. Figure 5 demonstrates that the CG KI mice and Fmr1 KO mice show a relatively linear progression in deficits as a function of CGG repeat length toward the Fragile X Full Mutation range for spatial and temporal processing. However, the data also suggest that the Fmr1 KO mouse demonstrates an unpredicted general memory deficit in the novel object detection task that was not shared with the CGG KI mouse models.

Figure 5. Spatial and temporal processing deficits in CGG KI and Fmr1 KO mice scale with CGG repeat length.

A. Metric or Coordinate task. B. Topological or Categorical task. C. Temporal Ordering for Visual Objects task. D. Novelty Detection for Visual Objects task. Data from Hunsaker et al., 2012; King and Jope, 2013; and Franklin et al., 2013^44,47,48.

Using entirely different groups of mice, a group in Rotterdam developed an automated version of the ladder rung task called the Erasmus ladder, upon which the testing and collection of data are fully automated^53–55. Similar to the ladder rung apparatus described above, the Erasmus ladder has a series of horizontal ladder-steps that the mouse must traverse in order to enter an escape/start box at the end. The apparatus was designed to use puffs of air to induce the mouse to shuttle between the two boxes by crossing the ladder steps. The system automatically captures step times, foot slips, misplacement of limbs, etc. Cupido⁵³ found that CGG KI mice showed a significantly increased number of foot slips and missteps (Figure 6), although they did not differ from wildtype mice in step time, suggesting intact general motor function. Additionally, the CGG KI mice were able to perform the motor associative learning task as well as wild type litter mate control animals. In contrast, Fmr1 KO mice showed a deficit for the associative learning task, never performing as well as CGG KI and wildtype control animals; however, the number of footslips and missteps did not differ from wildtype controls, suggesting intact visuospatial, visuomotor, and basic motor functions.

Figure 6. Data redrawn from Cupido (2009)⁵³ demonstrating the dissociation between motor performance and motor associative learning in CGG KI mice and the Fmr1 KO mouse model of Fragile X Syndrome.

Note the dissociation between motor endophenotypes in the CGG KI and Fmr1 KO Mice. All CGG KI mice were from the High CGG repeat group in other figures.

It has been demonstrated previously that visuomotor function depends on spatiotemporal integration^40,43. This means that for an individual to act in space, he must be able to (1) select a goal of action and (2) plan the movements, (3) initiate the movements, (4) modify the ongoing movement using updated spatiotemporal information, and (5) properly terminate the movement as intended. Intact spatiotemporal updating prevents misguided movements, such as reaching for a cup and knocking it off the table because an inability to stop or slow the progress of the hand. This is increasingly important if reaching or acting in space extends to processing angles among stimuli or targets that change over time. This updating process is what may be deficient in Fragile X Premutation carriers and in FXTAS patients, and in the CGG KI mice as they show similar spatiotemporal processing deficits and thus reduced spatial and temporal resolution. This type of spatiotemporal deficit may underlie anecdotal reports of subclinical apraxia and general clumsiness among Premutation carriers.

Similar to the novel object detection task, it was clear from the data that the Fmr1 KO mice demonstrate a deficit in motor learning that was not present in the CGG KI mouse model. The clear dissociation between motor performance (or visuospatial function) deficits in the CGG KI mouse in the absence of motor learning deficits and the inverse case in the Fmr1 KO mice provides a unique opportunity to evaluate separate components of the motor system in these models. Such data are critical to inform research into the differences between motor phenotypes in Fragile X Premutation carriers with FXTAS and Full Mutation carriers with Fragile X Syndrome.

Analytical verification. As previously demonstrated⁴⁴, an unsupervised hierarchal clustering algorithm can correctly sort the CGG KI mouse performance on a number of behavioral tasks into appropriate repeat-length subgroups. To verify that these observations comprised a behavioral endophenotype, a similar clustering analysis was performed on a dataset consisting of the CGG KI mice⁴⁴ and the Fmr1 KO mice^47,48. Only the metric or coordinate task, the topological or categorical task, the temporal ordering and novelty detection are concluded in this analysis as the number of animals in each group were insufficient in the other experiments reported in the review to run this analysis. In all cases, similar to Hunsaker et al. (2012)⁴⁴, support vector machines (SVM), a supervised machine learning technique, were performed to determine whether patterns in the behavioral data could be interpreted as indications of the expansion of CGG repeat length into the Fragile X Full Mutation. To assess the performance of the SVM classifier, iterative k-fold cross validation (10-fold, 5-fold, 3-fold, and leave-one-out cross validation) methods were used to estimate the accuracy of the classifier that predicts the group classification of a test sample. In the figures, the wildtype mice were removed from the dataset and the analyses were repeated to verify that CGG KI and Fmr1 KO mice could be correctly classified by CGG repeat length (i.e., Low CGG repeat vs. High CGG repeat vs. Fmr1 KO).

Figure 7 is a heatmap representation with all the behavioral tasks included and Figure 8 is a similar heatmap with the novel object detection task removed to evaluate how well the algorithm could sort the Fmr1 KO mice and CGG KI mice without the benefit of the novel object detection task.

Figure 7. Heatmap visualizing unsupervised hierarchal clustering of CGG KI and Fmr1 KO mice based on overall behavioral performance.

Note how the algorithm was capable of separating groups with high levels of accuracy with only a single Fmr1 KO mouse and one High CGG KI mouse being misclassified. The colors along the right hand column of the heatmap correspond to the group colors used in Figure 5.

Figure 8. Heatmap visualizing unsupervised hierarchal clustering of CGG KI and Fmr1 KO mice based on overall behavioral performance.

Note how the algorithm was capable of separating groups with high levels of accuracy, with only a single high CGG KI mouse being misclassified. The colors along the right hand column of the heatmap correspond to the group colors used in Figure 5.

Male and female Fragile X Premutation carriers

Human Premutation carriers. Initially, because of the presumed protective effect of a second FMR1 gene on a non-mutated X chromosome, it was assumed that female Fragile X Premutation carriers would not show similar levels of histopathological features (e.g., intranuclear inclusions) as male Premutation carriers⁵⁶.

In 2012, a study was undertaken to specifically determine if this assumption was correct²⁰. Specifically, the same methods as employed earlier^57–59 were replicated using sections from female post mortem tissues from Fragile X Premutation carriers. The most intriguing result from this study was that the female Premutation carriers did not in fact have extremely low numbers of intranuclear inclusions in neurons.

The number of intranuclear inclusions in neurons was virtually identical to that observed in male Premutation carrier cases. However, these female cases demonstrated a clear reduction in the number of intranuclear inclusions in astroglia. This reduction, furthermore, seemed to be inversely related to activation ratio, or directly related to the proportion of premutation FMR1 expressed in brain. This overall effect does suggest an overall reduction in histopathological features in female compared to male Premutation carriers, but not a clear cut one²⁰.

An additional datum of interest from this study was that one of the female Premutation carrier cases never developed motor features congruent with a FXTAS diagnosis. In fact, this case was included initially in the sample to demonstrate that the presence of intranuclear inclusions is associated with FXTAS. In other words, it was hypothesized that this case would be either free of inclusions or else have at best very low numbers of these pathologic anatomical features. This case also had a very high activation ratio, meaning the mutated FMR1 gene was only expressed in 20% of their cells. Despite these presumed protective factors and lack of motor phenotypes during life, this case showed the highest number of intranuclear inclusions in the study-suggesting that the inclusion bodies appear to be related to the Fragile X Premutation itself, not to the clinical manifestations of genetic disease (e.g., FXTAS).

CGG KI mouse model. In the CGG KI mouse model a parallel experiment was performed with the female Premutation carrier study described above. What was observed was striking: there was a 20–30% reduction in the number of inclusion bodies in neurons of female CGG KI mice when compared to male CGG KI mice with the same CGG repeat length. For this pathology in astroglia, the female CGG KI mice showed a clear and reliable 50% reduction in intranuclear inclusions compared to the male CGG KI mice with the same CGG repeat length^60,61.

These findings suggest there is a clear dosage effect, at least in mice, for the presence of intranuclear inclusions in CGG KI mice. These data strongly suggest that Fmr1 expression levels play an as yet undefined role in inclusion body formation.

Fragile X Premutation and Fragile X Full mutation carriers

Human Premutation and Full Mutation carriers. In 2011, it was discovered that Fragile X Full Mutation carriers counter intuitively show the hallmark neuropathological feature of The Fragile X Premutation and FXTAS: eosinophilic intranuclear inclusions that stain positive for ubiquitin^62,63. To identify how this was possible, given that earlier analyses had suggested that these cases lacked FMR1 mRNA and FMRP expression, further, more sensitive, molecular analyses were performed. These analyses discovered that FMR1 mRNA and FMRP were in fact expressed, just at very low levels that were missed by previous techniques^62,63. All said and done, these Fragile X Full Mutation carriers showed approximately 1–2% of the total number of inclusions compared to Fragile X Premutation carriers analyzed in parallel. However, the number of inclusion bodies identified did seem to scale with the rather low expression levels of FMR1 mRNA and FMRP.

Subsequent studies have borne this hypothesis out by evaluating neuropathologic anatomical feature in individuals that have a size mosaic for the Fragile X Premutation and Full Mutation (i.e., they express alleles with Premutation and alleles with Full Mutation length mutations). In one particular case, the expression of histopathological features again scaled with the expression levels of FMR1 mRNA and FMRP in brain. Interestingly, this patient had been diagnosed with Fragile X Syndrome and Parkinsonism during their lifetime, but upon the identification of these neuropathologic features and a re-evaluation of the medical records suggested that this individual had both Fragile X Syndrome as well as FXTAS, despite the fact that this comorbidity had previously been discounted as theoretically impossible⁶⁴.

CGG KI mouse model. In 2012 it was determined that there was a somewhat linear relationship between CGG repeat length and the quantity of intranuclear inclusions in a number of brain areas in the CGG KI mouse⁶⁰. The similarity with the human cases carrying the Fragile X Full Mutation described above is that mice carrying a mutation upwards of 350 CGG repeats in length were evaluated. Similar to the cases described above, as CGG KI mice show CGG repeat lengths beyond 230 repeats in length, the number of intranuclear inclusions reduces dramatically. This parallels research done in the same mouse line in Rotterdam⁷³ that suggested Fmr1 mRNA levels and FMRP levels begin to reduce as they approach 200+ CGG repeats.

Unfortunately to date, no matter how long the CGG repeat length becomes in CGG KI mice, the gene does not hypermethylate and silence in the same way as the human FMR1 gene. This means that at present there is no clear and unambiguous comparison between human and mouse Full Mutation alleles, so far as the analysis of histopathological features associated with disease states is concerned.

Behavioral endophenotype

Overall, there are two clear conclusions we can draw from the data described in this review. The first is that the behavioral phenotypes of CGG KI mice and Fmr1 KO mice appear to be related in some way. This suggests that there is a behavioral spectrum upon which these two mouse models can be found³⁸. Intriguingly, early models positing a spatial and temporal processing deficit model for Fragile X Syndrome and the Fragile X Premutation now have direct experimental support⁶⁵. We confirmed this by demonstrating that an unsupervised classifier is capable of using the behavioral data from this rapid battery to identify and sort the CGG KI mice into repeat length groups that are distinct from Fmr1 KO mice, which are reliably sorted as well. These behavioral and analytical results suggest that spatiotemporal processing may be an analytically powerful outcome measure that can be used as an endpoint for interventional, therapeutic, or diagnostic experiments.

However, there is also a clear additional behavioral deficit associated with having the Fragile X Full Mutation that is not present in the Fragile X Premutation. This was best observed in the novel object detection task, wherein the CGG KI mice displayed no deficits whatsoever, whereas the Fmr1 KO mice showed a rather profound inability to perform the task. The same type of dissociation exists within the motor domain. CGG KI mice appear to show motor performance deficits, possible from impaired visuomotor function but intact motor learning, whereas the Fmr1 KO mouse shows intact motor function, but impaired motor associative learning. Although these data appear incongruous, the mouse models do actually appropriately model the motor phenotypes associated in the Fragile X-Associated Disorders human population being modeled.

Together the behavioral results reviewed above demonstrating spatiotemporal and motor phenotypes in CGG KI and Fmr1 KO mice results suggest that the CGG KI and Fmr1 KO mouse models are valid models for evaluating any behavioral effects of the Fragile X Premutation and Full Mutation. This validity stems from the fact that populations of female Premutation carriers demonstrate similar deficits on spatiotemporal processing tasks as the CGG KI mice and Full Mutation carriers demonstrate clear spatiotemporal deficits on standardized testing. Similarly, motor features in female Fragile X Premutation carriers without FXTAS related motor features have been described. Additionally, research involving Premutation carriers are identifying associations between behavioral performance and CGG repeat length, FMR1 mRNA expression levels, and FMRP expression levels; further supporting the use of the associated mouse models for preclinical experimentation.

Histopathological endophenotype

The second main conclusion is that the neuropathological features, although complicated, may also be post-mortem endophenotypes of the Fragile X Premutation and Full Mutation. In humans, it appears that there is a complicated but somewhat predictable relationship between expression of the FMR1 gene and the presence of intranuclear inclusions, at least within the range of the Fragile X Premutation. In the Full Mutation, it still appears that there is a relationship between FMR1 expression and pathological features, but this is complicated by the extremely low expression levels and similarly low numbers of intranuclear inclusion bodies^{20,57–64,66–69}.

In evaluating female and male Fragile X Premutation carriers, it becomes clear that there is an association between gene expression and histopathology-albeit again a complicated one. These results beg for a thorough analysis of cell-type specific FMR1 expression patterns and cell-type specific analyses of pathology.

Intriguingly, although an incomplete model lacking the Fmr1 promoter hypermethylation observed in human Fragile X Full Mutation carriers, the CGG KI mouse appears to recapitulate the results of studies carried out with human tissue. Since the CGG KI mouse model appears to model the histopathological features, it is a fair assumption that the CGG KI mouse should be useful to elucidate the process by which these intranuclear inclusions form in different cell types and potentially preclinically test potential therapies or interventions designed to modulate or manipulate the development of this pathology.

Limitations to the mouse models

Despite the fact that the CGG KI mouse appears to model the Fragile X Premutation relatively well, there are commonly overlooked weaknesses in the mice used to model the Fragile X Full Mutation that need to be overcome. The first of these difficulties is that at present there are not any good mouse models for FMR1 mutation mosaicism for either size or methylation status. This is important if we are to use the mouse model to adequately model the human condition, given that with increasing study, the genetics underlying Fragile X-Associated Disorders are revealed as more complicated than originally assumed. Additionally, none of the CGG KI mouse models show any evidence for Fmr1 promoter silencing, so none of the mice show the dramatic reduction in gene expression at the 200–230 CGG repeat threshold often described in human studies.

Although demonstrating similar pathology such as macroorchidism and altered dendritic development, the Fmr1 KO mouse model does not actually model the genetics of the Fragile X Full Mutation. The Fmr1 KO mouse was developed by inserting a NEO cassette into the first exon of the Fmr1 gene, thus silencing expression. However, although apparently a knockout, it was shown that this original Fmr1 KO mouse does express Fmr1 mRNA and FMRP, albeit nonfunctional and rapidly degraded. This is still the mouse the majority of researchers employ in their research. To eliminate this confound, a second generation of the Fmr1 KO mouse removed the promoter region entirely, thus completely silencing Fmr1 gene expression⁷⁴. The molecular results of these models are very different from hypermethylation virtually, but not absolutely, silencing transcription with the remaining mRNA and protein being normal in structure and completely functional. To fully model the Fragile X Full Mutation at a molecular level, it is necessary for models that show human-like methylation and expression patterns be developed.

Future directions

Moving forward, the mouse models associated with Fragile X-Associated Disorders (Fragile X Premutation and Full Mutation) will be an essential tool for informing human disease research as well as for preclinical interventional studies. This utility, however, will only occur so long as care is taken to specifically focus on the similarities with the human populations being studied.

For studying the development of neuropathological features, these mouse models may serve as testing platforms for early interventional studies. For the Fragile X Premutation, the CGG KI mouse can be used to determine if there is a critical point in development when an intervention may stave off the development of neuropathological features and potential neurodegeneration (i.e., progression toward a neurodegenerative course associated with FXTAS). Similarly, the Fmr1 KO mouse is already in use to determine if early intervention can reliably normalize development and dendritic architecture later in life in the absence of chronic treatment, thus improving outcome.

The CGG KI mouse model may further be useful to determine what potential factors contribute to the incomplete penetrance of FXTAS. In other words, one can test environmental toxicant exposure, dietary factors, rearing conditions, anxiety, stress, depression, or any number of factors or combinations of factors to determine if there is an environment × gene dosage × age interaction that underlies abnormal neurodevelopment and eventual neurodegeneration. Studies evaluating potential immune mediated pathology in the Fragile X Premutation are already being evaluated⁷⁵, particularly for any role of pro inflammatory cytokine levels in long term outcome.

Finally, from a purely behavioral standpoint, it is critical that task development continue for these mouse models that explicitly parallel the tasks being developed for humans and the cognitive and functional domains being emphasized in humans. The current paradigm of murine researchers testing compounds with a focus on prepulse inhibition and the water maze is outdated, not translatable to the clinic in any way, and only serves to stagnate the field. There are currently already a number of resources that provide a solution in the form of behavioral assays that can be creatively applied to model human neuropsychological task batteries ranging from those used currently to test Fragile X-Associated Disorders, the 22q11.2 Deletion Syndrome, and even the NIH Toolbox cognitive assessment tools (i.e., http://www.nihtoolbox.org). Only by thoroughly modeling the cognitive assays being used can any data using these models be clearly and validly interpreted^38,45,46.

With any luck, research using the CGG KI and Fmr1 KO mouse models of Fragile X-Associated Disorders will be able to provide critical information to researchers that can be readily translated to clinical applications. The need for rigorous study of these models is evidenced by the increasing frequency of small pharmaceutical companies developing compounds to treat Fragile X-Associated Disorders. However, to date these efforts have been less than successful in identifying clear hypotheses from the mouse models that can be easily translated into the clinic, much less into clinical trials.

Research into the CGG KI and Fmr1 KO mouse models and into Fragile X-Associated Disorders are at an impasse that only direct basic-clinical science translational research can overcome. It is only through clear communication across levels of science that the basic scientist and clinician can truly work together toward their common goals. It is only through these reciprocal interactions that innovation truly germinates, and the opportune moment to truly begin is now.