Robert Bowser

Mechanisms, models and biomarkers in amyotrophic lateral sclerosis

Abstract The last 30 years have seen a major advance in the understanding of the clinical and pathological heterogeneity of amyotrophic lateral sclerosis (ALS), and its overlap with frontotemporal dementia. Multiple, seemingly disparate biochemical pathways converge on a common clinical syndrome characterized by progressive loss of upper and lower motor neurons. Pathogenic themes in ALS include excitotoxicity, oxidative stress, mitochondrial dysfunction, neuroinflammation, altered energy metabolism, and most recently RNA mis-processing. The transgenic rodent, overexpressing mutant superoxide dismutase-1, is now only one of several models of ALS pathogenesis. The nematode, fruit fly and zebrafish all offer fresh insight, and the development of induced pluripotent stem cell-derived motor neurons holds promise for the screening of candidate therapeutics. The lack of useful biomarkers in ALS contributes to diagnostic delay, and the inability to stratify patients by prognosis may be an important factor in the failure of therapeutic trials. Biomarkers sensitive to disease activity might lessen reliance on clinical measures and survival as trial endpoints and reduce study length. Emerging proteomic markers of neuronal loss and glial activity in cerebrospinal fluid, a cortical signature derived from advanced structural and functional MRI, and the development of more sensitive measurements of lower motor neuron physiology are leading a new phase of biomarker-driven therapeutic discovery.

Biomarkers in amyotrophic lateral sclerosis: opportunities and limitations

Insights into the mechanisms of amyotrophic lateral sclerosis (ALS) have relied predominantly on the study of postmortem tissue. Modern technology has improved the ability of scientists to probe effectively the underlying biology of ALS by examination of genomic, proteomic and physiological changes in patients, as well as to monitor functional and structural changes in patients over the course of disease. While effective treatments for ALS are lacking, the discovery of biomarkers for this disease offers clinicians tools for rapid diagnosis, improved ways to monitor disease progression, and insights into the pathophysiology of sporadic ALS. The ultimate aim is to broaden the therapeutic options for patients with this disease.

Futsch/MAP1B mRNA Is a Translational Target of TDP-43 and Is Neuroprotective in a Drosophila Model of Amyotrophic Lateral Sclerosis

TDP-43 is an RNA-binding protein linked to amyotrophic lateral sclerosis (ALS) that is known to regulate the splicing, transport, and storage of specific mRNAs into stress granules. Although TDP-43 has been shown to interact with translation factors, its role in protein synthesis remains unclear, and no in vivo translation targets have been reported to date. Here we provide evidence that TDP-43 associates with futsch mRNA in a complex and regulates its expression at the neuromuscular junction (NMJ) in Drosophila. In the context of TDP-43-induced proteinopathy, there is a significant reduction of futsch mRNA at the NMJ compared with motor neuron cell bodies where we find higher levels of transcript compared with controls. TDP-43 also leads to a significant reduction in Futsch protein expression at the NMJ. Polysome fractionations coupled with quantitative PCR experiments indicate that TDP-43 leads to a futsch mRNA shift from actively translating polysomes to nontranslating ribonuclear protein particles, suggesting that in addition to its effect on localization, TDP-43 also regulates the translation of futsch mRNA. We also show that futsch overexpression is neuroprotective by extending life span, reducing TDP-43 aggregation, and suppressing ALS-like locomotor dysfunction as well as NMJ abnormalities linked to microtubule and synaptic stabilization. Furthermore, the localization of MAP1B, the mammalian homolog of Futsch, is altered in ALS spinal cords in a manner similar to our observations in Drosophila motor neurons. Together, our results suggest a microtubule-dependent mechanism in motor neuron disease caused by TDP-43-dependent alterations in futsch mRNA localization and translation in vivo.

Therapeutic neuroprotective agents for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal chronic neurodegenerative disease whose hallmark is proteinaceous, ubiquitinated, cytoplasmic inclusions in motor neurons and surrounding cells. Multiple mechanisms proposed as responsible for ALS pathogenesis include dysfunction of protein degradation, glutamate excitotoxicity, mitochondrial dysfunction, apoptosis, oxidative stress, and inflammation. It is therefore essential to gain a better understanding of the underlying disease etiology and search for neuroprotective agents that might delay disease onset, slow progression, prolong survival, and ultimately reduce the burden of disease. Because riluzole, the only Food and Drug Administration (FDA)-approved treatment, prolongs the ALS patient’s life by only 3xa0months, new therapeutic agents are urgently needed. In this review, we focus on studies of various small pharmacological compounds targeting the proposed pathogenic mechanisms of ALS and discuss their impact on disease progression.

Multicenter validation of CSF neurofilaments as diagnostic biomarkers for ALS

Abstract OBJECTIVE: Neurofilaments are leading neurochemical biomarkers for amyotrophic lateral sclerosis (ALS). Here, we investigated the effect of preanalytical factors on neurofilament concentrations in cerebrospinal fluid (CSF) in a “reverse” round-robin with 15 centers across Europe/U.S. METHODS: Samples from ALS and control patients (5/5 each center, nu2009=u2009150) were analyzed for phosphorylated neurofilament heavy chain (pNfH) and neurofilament light chain (NfL) at two laboratories. RESULTS: CSF pNfH was increased (pu2009<u20090.05) in ALS in 10 out of 15 centers and NfL in 5 out of 12 centers. The coefficient of variation (CV%) of pNfH measurements between laboratories was 18.7u2009±u200919.1%. We calculated a diagnostic cut-off of >568.5u2009pg/mL for pNfH (sensitivity 78.7%, specificity 93.3%) and >1,431pg/mL for NfL (sensitivity 79.0%, specificity 86.4%). CONCLUSION: Values in ALS patients are already comparable between most centers, supporting eventual implementation into clinical routine. However, continuous quality control programs will be necessary for inclusion in the diagnostic work-up.

Attenuated traumatic axonal injury and improved functional outcome after traumatic brain injury in mice lacking Sarm1

Axonal degeneration is a critical, early event in many acute and chronic neurological disorders. It has been consistently observed after traumatic brain injury, but whether axon degeneration is a driver of traumatic brain injury remains unclear. Molecular pathways underlying the pathology of traumatic brain injury have not been defined, and there is no efficacious treatment for traumatic brain injury. Here we show that mice lacking the mouse Toll receptor adaptor Sarm1 (sterile α/Armadillo/Toll-Interleukin receptor homology domain protein) gene, a key mediator of Wallerian degeneration, demonstrate multiple improved traumatic brain injury-associated phenotypes after injury in a closed-head mild traumatic brain injury model. Sarm1(-/-) mice developed fewer β-amyloid precursor protein aggregates in axons of the corpus callosum after traumatic brain injury as compared to Sarm1(+/+) mice. Furthermore, mice lacking Sarm1 had reduced plasma concentrations of the phophorylated axonal neurofilament subunit H, indicating that axonal integrity is maintained after traumatic brain injury. Strikingly, whereas wild-type mice exibited a number of behavioural deficits after traumatic brain injury, we observed a strong, early preservation of neurological function in Sarm1(-/-) animals. Finally, using in vivo proton magnetic resonance spectroscopy we found tissue signatures consistent with substantially preserved neuronal energy metabolism in Sarm1(-/-) mice compared to controls immediately following traumatic brain injury. Our results indicate that the SARM1-mediated prodegenerative pathway promotes pathogenesis in traumatic brain injury and suggest that anti-SARM1 therapeutics are a viable approach for preserving neurological function after traumatic brain injury.

Neuroprotection for Amyotrophic Lateral Sclerosis: Role of Stem Cells, Growth Factors, and Gene Therapy

Various molecular mechanisms including apoptosis, inflammation, oxidative stress, mitochondrial dysfunction and excitotoxicity have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), though the exact mechanisms have yet to be specified. Furthermore, the underlying restorative molecular mechanisms resulting in neuronal and/or non-neuronal regeneration have to be yet elucidated. Therapeutic agents targeting one or more of these mechanisms to combat either initiation or progression of the disease are under research. Novel treatments including stem cell therapy, growth factors, and gene therapy might prolong survival and delay progression of symptoms. Harnessing the regenerative potential of the central nervous system would be a novel approach for the treatment of motor neuron death resulting from ALS. Endogenous neural replacement, if augmented with administration of exogenous growth factors or with pharmaceuticals that increase the rate of neural progenitor formation, neural migration, and neural maturation could slow the rate of cell loss enough to result in clinical improvement. In this review, we discuss the impact of therapeutic treatment involving stem cell therapy, growth factors, gene therapy, and combination therapy on disease onset and progression of ALS. In addition, we summarize human clinical trials of stem cell therapy, growth factor therapy, and gene therapy in individuals with ALS.

Novel Death Defying Domain in Met entraps the active site of caspase-3 and blocks apoptosis in hepatocytes.

Met, the transmembrane tyrosine kinase receptor for hepatocyte growth factor (HGF), is known to function as a potent antiapoptotic mediator in normal and neoplastic cells. Herein we report that the intracellular cytoplasmic tail of Met has evolved to harbor a tandem pair of caspase‐3 cleavage sites, which bait, trap, and disable the active site of caspase‐3, thereby blocking the execution of apoptosis. We call this caspase‐3 cleavage motif the Death Defying Domain (DDD). This site consists of the following sequence: DNAD‐DEVD‐T (where the hyphens denote caspase cleavage sites). Through functional and mechanistic studies, we show that upon DDD cleavage by caspase‐3 the resulting DEVD‐T peptide acts as a competitive inhibitor and entraps the active site of caspase‐3 akin to DEVD‐CHO, which is a potent, synthetic inhibitor of caspase‐3 activity. By gain‐ and loss‐of‐function studies using restoration of DDD expression in DDD‐deficient hepatocytic cells, we found that both caspase‐3 sites in DDD are necessary for inhibition of caspase‐3 and promotion of cell survival. Employing mutagenesis studies, we show that DDD could operate independently of Mets enzymatic activity as determined by using kinase‐dead human Met mutant constructs. Studies of both human liver cancer tissues and cell lines uncovered that DDD cleavage and entrapment of caspase‐3 by DDD occur in vivo, further proving that this site has physiological and pathophysiological relevance. Conclusion: Met can directly inhibit caspase‐3 by way of a novel mechanism and promote hepatocyte survival. The results presented here will further our understanding of the mechanisms that control not only normal tissue homeostasis but also abnormal tissue growth such as cancer and degenerative diseases in which apoptotic caspases are at play. (Hepatology 2014;59:2010–2021)

Amyotrophic lateral sclerosis: Is the spinal fluid pathway involved in seeding and spread?

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder manifested primarily by loss of upper and lower motor neurons. Current explanations for disease progression invoke regional spread attributed to the transfer of pathogenic factors among physically contiguous neurons. However, this explanation incompletely explains certain clinical and in vitro data. Considering this, we propose that the cerebrospinal fluid (CSF) pathway is likely to be a key vector for seeding local and distal disease. Subsequent disease progression would be expected to occur independently via either axonal or CSF transmission. If one accepts the hypothesis that the CSF pathway is involved in ALS progression, it follows that the choroid plexus (CP) might well be a driver of the disease process. In support of this, we briefly review the anatomical and physiological features of the CSF pathway and the choroid plexus responsible for secreting CSF. In addition, we draw attention to the interface of the CP and CSF with the immune system. We then summarize both clinical and cell culture research that supports a key role of the CSF in the establishment and inter-neuronal spread of ALS, and which suggest directions for translational research.

ALS Associated Mutations in Matrin 3 Alter Protein-Protein Interactions and Impede mRNA Nuclear Export

Mutations in Matrin 3 have recently been linked to ALS, though the mechanism that induces disease in these patients is unknown. To define the protein interactome of wild-type and ALS-linked MATR3 mutations, we performed immunoprecipitation followed by mass spectrometry using NSC-34 cells expressing human wild-type or mutant Matrin 3. Gene ontology analysis identified a novel role for Matrin 3 in mRNA transport centered on proteins in the TRanscription and EXport (TREX) complex, known to function in mRNA biogenesis and nuclear export. ALS-linked mutations in Matrin 3 led to its re-distribution within the nucleus, decreased co-localization with endogenous Matrin 3 and increased co-localization with specific TREX components. Expression of disease-causing Matrin 3 mutations led to nuclear mRNA export defects of both global mRNA and more specifically the mRNA of TDP-43 and FUS. Our findings identify a potential pathogenic mechanism attributable to MATR3 mutations and further link cellular transport defects to ALS.