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.

Amyotrophic lateral sclerosis (ALS) is a deadly neurodegenerative disease characterized by loss of motor neurons, paralysis and eventual death. The mechanism of ALS is still incompletely understood, and the disease is to date without an effective remedy. Protein arginine deiminase 2 (PAD2) converts peptidyl-Arginine into peptidyl-Citrulline, a post-translational modification referred to as citrullination. Aberrant expression of PAD2 and protein citrullination are increased in several neurodegenerative conditions. Whether this increase is involved in ALS is unknown. In this study, we investigated PAD2 and protein citrullination in two genetic mouse models of ALS expressing human mutant SOD1G93A and PFN1C71G, respectively, and in post-mortem human ALS spinal cord. We show that the expression of PAD2 mRNA and protein expressions are increased progressively along with the ALS progression. Additionally, protein citrullination is increase following the same trend. These changes occur in areas with the most severe motor neuron degeneration including the spinal cord, and brainstem. We show that the increase in PAD2 and citrullinated proteins occur specifically in astrocytes coinciding with the development of reactive astrogliosis. Finally, we show that citrullinated proteins form non-astrocyte aggregate patterns; and are dominantly expressed in insoluble protein fractions. These results demonstrate that PAD2 dysregulation and increased protein citrullination are key characteristics of reactive astrogliosis, and possibly drive some type of protein aggregation in the pathogenesis of ALS. Because protein citrullination alters protein functions, our results suggest that PAD2 and protein citrullination play a role in astrogliosis and astrocytic toxicity in ALS and other neurodegenerative conditions.

The lack of methods to deliver transgene expression in spinal cord has hampered investigation of gene function and therapeutic targets for spinal cord diseases. Here, we report that a single intrathecal injection of recombinant adeno-associated virus rhesus-10 (rAAVrh10) into the lumbar cistern led to transgene expression in 60 to 90 % of the cells in the spinal cord. The transgene was expressed in all cell types, including neurons, glia, ependymal cells, and endothelial cells. Additionally, the transgene was expressed in some brain areas up to the frontal cortex and the olfactory bulb. The rAAV was distributed predominantly in the spinal cord, where its genome copy was over ten times that of the peripheral organs. Compared with intravenous injection, another method for rAAV delivery to the broad central nervous system (CNS), the intrathecal injection reduced the dosage of rAAV required to achieve similar or higher levels of transgene expression in the CNS by ~100-fold. Finally, the transduced areas were co-localized with the perivascular spaces of Virchow-Robin, from which the rAAV spreads further into the CNS parenchyma, thus suggesting that rAAV penetrated the CNS parenchyma through this pathway. Taken together, we have defined a fast and efficient method to deliver widespread transgene expression in mature spinal cord in mice. This method can be applied to stably overexpress or silence gene expression in the spinal cord to investigate gene functions in mammalian CNS. Additionally, this method can be applied to validate therapeutic targets for spinal cord diseases.

BACKGROUND: A proline-to-serine substitution at position-56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) causes a form of dominantly inherited motor neuron disease (MND), including typical and atypical amyotrophic lateral sclerosis (ALS) and a mild late-onset spinal muscular atrophy (SMA). VAPB is an integral endoplasmic reticulum (ER) protein and has been implicated in various cellular processes, including ER stress, the unfolded protein response (UPR) and Ca2+ homeostasis. However, it is unclear how the P56S mutation leads to neurodegeneration and muscle atrophy in patients. The formation of abnormal VAPB-positive inclusions by mutant VAPB suggests a possible toxic gain of function as an underlying mechanism. Furthermore, the amount of VAPB protein is reported to be reduced in sporadic ALS patients and mutant SOD1G93A mice, leading to the hypothesis that wild type VAPB plays a role in the pathogenesis of ALS without VAPB mutations. RESULTS: To investigate the pathogenic mechanism in vivo, we generated human wild type (wtVAPB) and mutant VAPB (muVAPB) transgenic mice that expressed the transgenes broadly in the CNS. We observed robust VAPB-positive aggregates in the spinal cord of muVAPB transgenic mice. However, we failed to find an impairment of motor function and motor neuron degeneration. We also did not detect any change in the endogenous VAPB level or evidence for induction of the unfolded protein response (UPR) and coaggregation of VAPA with muVAPB. Furthermore, we crossed these VAPB transgenic mice with mice that express mutant SOD1G93A and develop motor neuron degeneration. Overexpression of neither wtVAPB nor muVAPB modulated the protein aggregation and disease progression in the SOD1G93A mice. CONCLUSION: Overexpression of VAPBP56S mutant to approximately two-fold of the endogenous VAPB in mouse spinal cord produced abundant VAPB aggregates but was not sufficient to cause motor dysfunction or motor neuron degeneration. Furthermore, overexpression of either muVAPB or wtVAPB does not modulate the course of ALS in SOD1G93A mice. These results suggest that changes in wild type VAPB do not play a significant role in ALS cases that are not caused by VAPB mutations. Furthermore, these results suggest that muVAPB aggregates are innocuous and do not cause motor neuron degeneration by a gain-of-toxicity, and therefore, a loss of function may be the underlying mechanism.