Increased protein citrullination (PC) and dysregulated protein arginine deiminase (PAD) activity have been observed in several neurodegenerative diseases. PC is a posttranslational modification catalyzed by the PADs. PC converts peptidyl-arginine to peptidyl-citrulline, thereby reducing the positive charges and altering structure and function of proteins. Of the five PADs, PAD2 is the dominant isoform in the central nervous system (CNS). Abnormal PC and PAD dysregulation are associated with numerous pathological conditions, including inflammatory diseases and neurodegeneration. Animal model studies have shown therapeutic efficacy from inhibition of PADs, thus suggesting a role of PC in pathogenesis. To determine whether PC contribute to amyotrophic lateral sclerosis (ALS), a deadly neurodegenerative disease characterized by loss of motor neurons, paralysis, and eventual death, we investigated alterations of PC and PAD2 in two different transgenic mouse models of ALS expressing human mutant SOD1G93A and PFN1C71G, respectively. PC and PAD2 expression are altered dynamically in the spinal cord during disease progression in both models. PC and PAD2 increase progressively in astrocytes with the development of reactive astrogliosis, while decreasing in neurons. Importantly, in the spinal cord white matter, PC accumulates in protein aggregates that contain the myelin proteins PLP and MBP. PC also accumulates progressively in insoluble protein fractions during disease progression. Finally, increased PC and PAD2 expression spatially correlate with areas of the CNS with the most severe motor neuron degeneration. These results suggest that altered PC is an integral part of the neurodegenerative process and potential biomarkers for disease progression in ALS. Moreover, increased PC may contribute to disease-associated processes such as myelin protein aggregation, myelin degeneration, and astrogliosis.

Modestly increased expression of transactive response DNA binding protein (TDP-43) gene have been reported in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neuromuscular diseases. However, whether this modest elevation triggers neurodegeneration is not known. Although high levels of TDP-43 overexpression have been modeled in mice and shown to cause early death, models with low-level overexpression that mimic the human condition have not been established. In this study, transgenic mice overexpressing wild type TDP-43 at less than 60% above the endogenous CNS levels were constructed, and their phenotypes analyzed by a variety of techniques, including biochemical, molecular, histological, behavioral techniques and electromyography. The TDP-43 transgene was expressed in neurons, astrocytes, and oligodendrocytes in the cortex and predominantly in astrocytes and oligodendrocytes in the spinal cord. The mice developed a reproducible progressive weakness ending in paralysis in mid-life. Detailed analysis showed ∼30% loss of large pyramidal neurons in the layer V motor cortex; in the spinal cord, severe demyelination was accompanied by oligodendrocyte injury, protein aggregation, astrogliosis and microgliosis, and elevation of neuroinflammation. Surprisingly, there was no loss of lower motor neurons in the lumbar spinal cord despite the complete paralysis of the hindlimbs. However, denervation was detected at the neuromuscular junction. These results demonstrate that low-level TDP-43 overexpression can cause diverse aspects of ALS, including late-onset and progressive motor dysfunction, neuroinflammation, and neurodegeneration. Our findings suggest that persistent modest elevations in TDP-43 expression can lead to ALS and other neurological disorders involving TDP-43 proteinopathy. Because of the predictable and progressive clinical paralytic phenotype, this transgenic mouse model will be useful in preclinical trial of therapeutics targeting neurological disorders associated with elevated levels of TDP-43.

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease is the most common form of motor neuron disease. In familial ALS, Multiple mutations of, PFN1 gene a well-known actin-binding protein have been linked to ALS disease recently. Phosphorylation in many degenerative conditions plays an important role in disease mechanism but its potential role in ALS remains not fully understood. We sought to look further into not previously studied phosphorylation of PFN1 as a potential contributor to aggregation and toxicity in ALS. Using different histochemistry and cytochemistry and molecular biology approaches, we observed that phosphorylation on Profilin shows a very distinctive pattern in PFN1C71G andSOD1G93A disease models. This modification is abundantly found in both astrocytes and white matter which latter indeed marks a staining pattern that is indistinguishable between two ALS mice model compared to controls. Interestingly, pPFN1 reactive areas colocalized with Myelin in the spinal cord are frequently found in the proximity of CD68 positive macrophages. Moreover, biochemical fractionation using ultracentrifugation detects endogenous pPFN1 in the highly insoluble fraction of protein lysate from both PFN1C71G andSOD1G93A model. Finally, a similar staining pattern to the ALS mice model was also observed in human sporadic ALS cases. Overall, our results suggest for the first time a role for phosphorylation of PFN1 in protein aggregation and white matter pathology in ALS that will shed more light on the mechanism of disease and developing potential therapeutics in near future.