Jesse R. McLean

Isoform-specific antibodies reveal distinct subcellular localizations of C9orf72 in amyotrophic lateral sclerosis.

A noncoding hexanucleotide repeat expansion in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). It has been reported that the repeat expansion causes a downregulation of C9orf72 transcripts, suggesting that haploinsufficiency may contribute to disease pathogenesis. Two protein isoforms are generated from three alternatively spliced transcripts of C9orf72; a long form (C9‐L) and a short form (C9‐S), and their function(s) are largely unknown owing to lack of specific antibodies.

An aggregate-inducing peripherin isoform generated through intron retention is upregulated in amyotrophic lateral sclerosis and associated with disease pathology.

The neuronal intermediate filament protein peripherin is a component of ubiquitinated inclusions and of axonal spheroids in amyotrophic lateral sclerosis (ALS). Overexpression of peripherin causes motor neuron degeneration in transgenic mice and variations within the peripherin gene have been identified in ALS cases. We have shown previously the abnormal expression of a neurotoxic peripherin splice variant in transgenic mice expressing mutant superoxide dismutase-1. These findings indicated that abnormalities of peripherin splicing may occur in ALS. In the current study, peripherin splice variants were identified by reverse transcription-PCR of human neuronal RNA and comparisons in expression made between control and ALS spinal cord using Western blot analysis and immunocytochemistry. Using this approach we have identified a novel peripherin transcript retaining introns 3 and 4 that results in a 28 kDa splice isoform, designated Per 28. Using an antibody specific to Per 28, we show that this isoform is expressed at low stoichiometric levels from the peripherin gene, however causes peripherin aggregation when its expression is upregulated. Importantly we show an upregulation of Per 28 expression in ALS compared with controls, at both the mRNA and protein levels, and that Per 28 is associated with disease pathology, specifically round inclusions. These findings are the first to establish that peripherin splicing abnormalities occur in ALS, generating aggregation-prone splice isoforms.

The complement factor C5a receptor is upregulated in NFL-/- mouse motor neurons.

In NFL-/- mice, a model of motor neuron degeneration in ALS, degenerating spinal motor neurons express high levels of the receptor for the C5a anaphylatoxin (C5aR) early in the disease process. C5a is a potent in vitro neurotoxin for both Neuro2A and NGF-differentiated PC12 cells. While no interaction was observed between glutamate and C5a, both C5a and kainate upregulated the expression of activated C5aR. C5aR expression was increased in motor neurons in ALS. This data suggests that the early upregulation of C5aR may contribute to motor neuron damage that potentiates excitotoxicity in ALS.

A novel peripherin isoform generated by alternative translation is required for normal filament network formation.

J. Neurochem. (2008) 104, 1663–1673.

Temporal profiles of neuronal degeneration, glial proliferation, and cell death in hNFL(+/+) and NFL(−/−)mice

Neurofilament (NF) aggregate formation within motor neurons is a pathological hallmark of both the sporadic and familial forms of amyotrophic lateral sclerosis (ALS). The relationship between aggregate formation and both microglial and astrocytic proliferation, as well as additional neuropathological features of ALS, is unknown. To examine this, we have used transgenic mice that develop NF aggregates, through either a lack of the low‐molecular‐weight NF subunit [NFL (−/−)] or the overexpression of human NFL [hNFL (+/+)]. Transgenic and wild‐type C57bl/6 mice were examined from 1 month to 18 months of age, and the temporal pattern of motor neuron degeneration, microglial and astrocytic proliferation, and heat shock protein‐70 (HSP‐70) expression characterized. We observed three overlapping phases in both transgenic mice, including transient aggregate formation, reactive microgliosis, and progressive motor neuron loss. However, only NFL (−/−) mice demonstrated significant astrogliosis and HSP‐70 upregulation in both motor neurons and astrocytes. These in vivo models suggest that the development of NF aggregates in motor neurons leads to motor neuron death, but that the interaction between the degenerating motor neurons and the adjacent non‐neuronal cells may differ significantly depending on the etiology of the NF aggregate itself.

Distinct biochemical signatures characterize peripherin isoform expression in both traumatic neuronal injury and motor neuron disease

J. Neurochem. (2010) 114, 1177–1192.

A two-hybrid screen identifies an unconventional role for the intermediate filament peripherin in regulating the subcellular distribution of the SNAP25-interacting protein, SIP30.

Peripherin is a type III intermediate filament protein, the expression of which is associated with the acquisition and maintenance of a terminally differentiated neuronal phenotype. Peripherin up‐regulation occurs during acute neuronal injury and in degenerating motor neurons of amyotrophic lateral sclerosis. The functional role(s) of peripherin during normal, injurious, and disease conditions remains unknown, but may be related to differential expression of spliced isoforms. To better understand peripherin function, we performed a yeast two‐hybrid screen on a mouse brain cDNA library using an assembly incompetent peripherin isoform, Per‐61, as bait. We identified new peripherin interactors with roles in vesicular trafficking, signal transduction, DNA/RNA processing, protein folding, and mitochondrial metabolism. We focused on the interaction of Per‐61 and the constitutive isoform, Per‐58, with SNAP25 interacting protein 30 (SIP30), a neuronal protein involved in SNAP receptor‐dependent exocytosis. We found that peripherin and SIP30 interacted through coiled‐coil domains and colocalized in cytoplasmic aggregates in SW13vim(−) cells. Interestingly, Per‐61 and Per‐58 differentially altered the subcellular distribution of SIP30 and SNAP25 in primary motor neurons. Our findings suggest a novel role of peripherin in vesicle trafficking.

Isoform-specific expression and ratio changes accompany oxidant-induced peripherin aggregation in a neuroblastoma cell line

The type III intermediate filament peripherin is found associated with pathological inclusions present within motor neurons of patients with amyotrophic lateral sclerosis (ALS). Peripherin intra-isoform associations contribute to filament network formation at defined stoichiometric ratios. Distinct biochemical signatures characterize peripherin isoform expression in traumatic neuronal injury and motor neuron disease, while disruptions to peripherin alternative splicing or translation are associated with inclusion formation. In our efforts to identify pathological relationships between peripherin isoform expression and inclusion formation, we provide evidence of peripherin isoform-specific expression and ratio changes with concomitant, dose-dependent inclusion formation in response to oxidative stress. Upon increasing exposure to physiologically relevant levels of hydrogen peroxide in Neuro-2a cells, we observed a significant increase and decrease in peripherin isoforms Per-58 and Per-45, respectively, with peripherin-specific perikaryal aggregation of filaments 10-15 μm in diameter. Interestingly, peripherin-immunoreactive inclusions showed no overt carbonylation, suggesting that aggregation may serve a physiologically relevant role during oxidative stress. These findings provide novel insight into the biological significance of peripherin isoforms and inclusion formation, with relevance to the pathology of ALS.