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Familial ALS

Identification of Genetic Factors Contributing to ALS

The identification of the genetic causes of ALS is necessary to further knowledge of the cellular pathways involved in neurodegeneration and contribute towards establishing targets for therapeutic intervention. Landers laboratory has a proven history of identify genes contributing to familial ALS (FALS) using a variety of approaches. Lab efforts were instrumental towards the identification of FUS as a causative gene for FALS using a combination of linkage analysis, homozygosity mapping and high-throughput Sanger sequencing. Through Dr. Landers long-standing interest of novel technologies, Landers laboratory employed exome capture and next-generation sequencing to identify mutations in the profilin 1 (PFN1) as a cause of FALS.

Dr. Landers has developed extensive collaborations with the leaders of ALS genetic research. As a result, Landers laboratory now contains the largest collection of FALS exome data in existence consisting of 1,376 FALS derived from 11 countries. Recently, Landers Lab has focused on using rare variant burden analysis (RVB) of exome sequencing results on this cohort to identify novel genes for FALS. In brief, RVB compares the combined frequency of rare variants within all genes in a case-control cohort. Candidate associations are identified by significant differences after multiple test correction. Through our efforts, we identified mutations in the TUBA4A gene, which encodes the Tubulin, Alpha 4A protein, are associated with FALS. To my best knowledge, this published study represents the first successful application of rare variant burden analysis for the discovery of a novel disease gene.

Currently, my laboratory continues to work towards the identification of additional novel ALS genes through various approaches and taking full advantage of our collection of FALS exome data. This includes further expanding this cohort and developing novel methodologies based on RVB. Most recently, we have improved RVB by developing a training session for the analysis with known ALS genes. This innovative method had led us to the identification of a novel risk factor that is observed in ~3% of ALS patients. The manuscript of this discovery is under review.

Overall, our efforts have focused primarily on using exome sequencing to identify genetic factors contributing to ALS. Although, this approach has provided a wealth of information, it is clearly not comprehensive. As demonstrated by numerous faculty members at our university, non-coding regions of the genome are exceedingly important for the regulation of various cellular processes. Based on these findings, and the dramatic reduction in price, we have begun the process of whole genome sequencing (WGS) our extensive FALS cohort. We anticipate that the results of this WGS will indeed reveal novel non-coding regions of the genome contributing to ALS.

Lastly, my lab has become more focused recently in the genetic factors contributing to sporadic ALS (SALS). In contrast to FALS, SALS cases represents the vast majority of all ALS cases (~90%). Based on the lack of a family history and supported by heritability studies, the genetic contribution of SALS is lower compared to FALS. As a result, it presents a difficult challenge to identify genetic factors contributing to SALS. In fact, it is anticipated that the identification of rare risk factors for SALS will require the WGS of tens of thousands of cases and controls. Such an endeavor is well beyond the capabilities (scientifically and financially) of a single laboratory. As a result of this limitation, the ALS scientific community has created a worldwide consortium called Project MinE. The goal of Project MinE is to WGS the DNA from 15,000 ALS cases and 7,500 controls. Currently 16 countries have joined Project MinE with more to follow. In October 2014, I was appointed as co-Director of the Project MinE effort in the USA (Project MinE USA), along with Dr. Jonathan Glass of Emory University. Our efforts have thus far resulted in the WGS of nearly 600 samples and we anticipate another 800 samples within the next year. Currently, Project MinE has reached nearly one-third of its goal of 22,500 WGS in total. Again, it is our hope that the findings from this effort will further help us to further understand the pathogenesis of ALS.  

It is anticipated that the investigation of ALS genetics will continue to be major focus of my laboratory for the foreseeable future. There are still many unexplored areas of ALS genetics and I anticipated that we will be successful in our endeavors. Such areas will include the role of non-coding regions, indels, copy number variations, genetic/environment interactions and epigenetic factors in the pathogenesis of ALS. Given the complexity of the future ALS genetic efforts, many of these genetic projects will likely be performed in continued collaboration with other leaders in ALS genetics. Project MinE is a prime example of such collaborative effort.

Establishing the Contribution of Cytoskeletal Defects in ALS

Determining the pathways contributing to ALS pathogenesis is essential for identifying novel targets for therapeutic intervention. Through our genetics efforts described above, we have identified two genes associated with ALS that are both tightly linked to cytoskeletal stability and dynamics, PFN1 and TUBA4A. PFN1 is crucial for the conversion of monomeric (G)-actin to filamentous (F)-actin. Our efforts have shown that mutations lead to destabilized, insoluble aggregates. Additionally, mutations in PFN1 decrease actin bound levels, inhibit axon outgrowth, decrease growth cone size and display a reduced F/G-actin ratio. Similarly, TUBA4A mutants result in altered microtubule polymerization, a destabilized microtubule network, and altered microtubule dynamics. Cytoskeletal alterations are also a common pathological feature in ALS patients. These studies, as well as those of several others, establish that cytoskeletal defects are a major pathway contributing to ALS pathogenesis. A major focus of our laboratory is to further dissect how such alterations lead to disease pathogenesis. Towards this goal, we are currently embarking on several differing research approaches. One such approach is the development of model organisms.

The development of model organisms has proven to be tremendous resource for the study of human diseases. Towards this end, my laboratory has focused on the development of murine models of ALS based on our identification of mutations in PFN1 and TUBA4A. In collaboration with Dr. Zuoshang Xu at UMass, we have recently completed the development of a transgenic mouse that expresses mutant PFN1. Interestingly, this model demonstrates several of the characteristics of ALS, including late onset progressive paralysis, decreased weight, decreased grip strength and motor neuron loss. To date, only the mutant SOD1 mouse model recapitulates the ALS phenotype observed in humans more faithfully than this mutant PFN1 mouse model. Additionally, in collaboration with Jackson Laboratory, we have developed mice that harbor point mutations in TUBA4A using CRIPR technology. Currently, 5 different TUBA4A mutations have been introduced into mice. Experiments are currently underway to compare mice that heterozygous or homozygous for TUBA4A mutations to wild-type mice to determine whether they develop traits similar to ALS. Although the introduction of point mutations in TUBA4A represents the best mouse model, it is conceivable that such mice may not develop an ALS phenotype or possibly display symptoms at a mild level with a long survival rate. As such, difficulties may arise in using such models in certain experimental situations. Furthermore, all ALS models based on gene mutations to date have required overexpression of a transgene to produce a representative phenotype. Based on this rational, we are also developing transgenic mouse models overexpressing either wild-type or mutant TUBA4A proteins in collaboration with Jackson Laboratories. Undoubtedly, these mouse models have opened up several new avenues of research to further understand the pathogenesis of ALS with a focus on cytoskeletal defects. These include the studying disease development and progression, changes in gene expression over time and examining how cytoskeletal defects lead to alterations in other cellular processes. As such, I anticipate that the study of these models will be a major focus of my laboratory to study cytoskeletal defects in ALS for the foreseeable future. 

Development of Therapeutic Treatments for ALS

The ultimate goal of disease research is the development of novel or improved therapeutic treatments. Currently, riluzole represents the only therapeutic treatment for ALS patients. Unfortunately, this treatment only results in a minimal increase in the survival for ALS patients (3-6 months). As described above, alterations to cytoskeletal structure and dynamics observed in ALS patients disrupt essential cellular functions which are necessary for the maintenance of motor neurons. Based on these observations, my laboratory has recently begun to focus on identifying potential therapeutic targets that can rescue relevant disease phenotypes by regulating cytoskeletal function. Towards this end, our group is leading a multi-center effort to identify therapeutic targets for cytoskeletal defects in ALS. In particular, the laboratory of Dr. Steve Finkbeiner (UCSF) is using primary rodent neurons that express mutant TUBA4A or PFN1 as a model of ALS-related cytoskeletal defects. A portion of a siRNA library focused on cytoskeletal genes was screened to identify modifiers that can rescue phenotypic defects (survival and neurite extension) observed in these cells. Identified hits will be tested in vitro and in vivo models by the laboratories of Dr. Bruce Goode (Brandeis University), Dr. Daryl Bosco (UMass Medical School) and myself to characterize their benefit, mechanism of action and influence on all forms of ALS. The initial screening of the siRNA library has recently been completed. Interestingly, the top hit in the primary screen for the rescue of mutant TUBA4A phenotype, not only rescued phenotypic defects in mutant PFN1 expressing neurons but also defects in neurons expressing mutant TDP-43, an ALS-associated gene with no direct link to cytoskeletal regulation. Testing of the beneficial effects of this siRNA in additional assays has begun.

Although this current screening approach to identify novel therapeutic targets has merit, it is restricted in its application. In particular, the use of primary rodent neurons is expensive and time-consuming thus limiting number of molecules that can be screened by this approach. As an alterative, my laboratory has begun to develop two inexpensive high-throughput assays that are based on rescue defects in microtubule dynamics and stability observed mutant TUBA4A expressing cells. The first assay is based on our observation that in cells expressing mutant TUBA4A, the unstable cytoskeleton can be easily washed away with a low detergent solution whereas in cells expressing wild-type TUBA4A, the cytoskeleton remains intact. As such, by fluorescently labelling the cytoskeleton, we can screen for compounds that stabilize the cytoskeleton and thus rescue this phenotype. The second assay is based on the microtubule plus-end protein EB3. Cells expressing GFP-EB3 can be subject to live-cell imaging allowing individual microtubules to be visualized and analysed to determine whether they are undergoing polymerization, catastrophe, rescue or stalling. By screening cells expressing mutant TUBA4A and GFP-EB3, we can screen for compounds that can rescue the defects in microtubule dynamics. Batch image processing and analysis will be accomplished through developing MATLAB scripts in conjunction with the UMass High Performance Computing Cluster (regularly used by our group for the analysis of deep sequencing data). We intend to use these assays to screen compounds at the Small Molecule Screening Facility at UMass and are currently working with Drs. Paul Thompson and Sangram Parelkar to optimize these assays to the fullest. 

In both studies, the resulting hits can be testing in numerous assays to establish their beneficial effect. As described above, our intent is to use the mutations in TUBA4A and PFN1 strictly as models for the cytoskeletal defects observed in ALS patients, yet, our goal is to identify therapeutics that can be applied to all forms of ALS. As such, we intend to test the beneficial effects of any therapeutic not only in mutant TUBA4A/PFN1 based assays but in assays based on mutation in non-cytoskeletal ALS related genes. In addition to cytoskeletal stability and dynamics, in vitro assays will examine axonal transport, neurite outgrowth, stress response and aggregate formation in primary motor neurons. These assays are well established in my laboratory. Therapeutics demonstrating the most promising results can then be tested in numerous ALS models commonly studied here at UMass. In addition to our PFN1 mouse model, other mouse models are based on the ALS associated genes these include the SOD1 model, the TDP-43 mouse model and the C9orf72 mouse model (recently developed by Dr. Robert Brown). Additional reagents for testing and characterizing potential therapeutics include iPS cells derived from numerous sporadic and familial ALS patients and several fly models based on alterations in C9orf72, SOD1, TDP-43, FUS and PFN1.