Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling.

In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3-stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.

Resistance to oncolytic myxoma virus therapy in nf1(-/-)/trp53(-/-) syngeneic mouse glioma models is independent of anti-viral type-I interferon.

Despite promising preclinical studies, oncolytic viral therapy for malignant gliomas has resulted in variable, but underwhelming results in clinical evaluations. Of concern are the low levels of tumour infection and viral replication within the tumour. This discrepancy between the laboratory and the clinic could result from the disparity of xenograft versus syngeneic models in determining in vivo viral infection, replication and treatment efficacy. Here we describe a panel of primary mouse glioma lines derived from Nf1 (+/-) Trp53 (+/-) mice in the C57Bl/6J background for use in the preclinical testing of the oncolytic virus Myxoma (MYXV). These lines show a range of susceptibility to MYXV replication in vitro, but all succumb to viral-mediated cell death. Two of these lines orthotopically grafted produced aggressive gliomas. Intracranial injection of MYXV failed to result in sustained viral replication or treatment efficacy, with minimal tumour infection that was completely resolved by 7 days post-infection. We hypothesized that the stromal production of Type-I interferons (IFNα/ß) could explain the resistance seen in these models; however, we found that neither the cell lines in vitro nor the tumours in vivo produce any IFNα/ß in response to MYXV infection. To confirm IFNα/ß did not play a role in this resistance, we ablated the ability of tumours to respond to IFNα/ß via IRF9 knockdown, and generated identical results. Our studies demonstrate that these syngeneic cell lines are relevant preclinical models for testing experimental glioma treatments, and show that IFNα/ß is not responsible for the MYXV treatment resistance seen in syngeneic glioma models.

A novel recessive Nefl mutation causes a severe, early-onset axonal neuropathy.

OBJECTIVE: To report the first cases of a homozygous recessive mutation in NEFL, the gene that encodes the light subunit of neurofilaments. METHODS: Clinical and electrophysiologic data were evaluated, and a sural nerve biopsy from one affected child was examined by immunohistochemistry and electron microscopy. The ability of the mutant protein to form filaments was characterized in an established cell culture system. RESULTS: Four of five siblings developed of a severe, progressive neuropathy beginning in early childhood. Serial nerve conduction studies showed progressively reduced amplitudes with age and pronounced slowing at all ages. Visual-evoked responses were slowed in three children, indicating that central nervous system axons were subclinically involved. All four affected children were homozygous for a nonsense mutation at glutamate 210 (E210X) in the NEFL gene; both parents were heterozygous carriers. A sural nerve biopsy from an affected patient showed markedly reduced numbers of myelinated axons; the remaining myelinated axons were small and lacked intermediate filaments. The E210X mutant protein did not form an intermediate filament network and did not interfere with the filament formation by wild-type human light subunit of neurofilaments in SW-13 vim(-) cells. INTERPRETATION: This is the first demonstration of a recessive NEFL mutation, which appears to cause a simple loss of function, resulting in a severe, early-onset axonal neuropathy with unique features. These results confirm that neurofilaments are the main determinant of axonal caliber and conduction velocity, and demonstrate for the first time that neurofilaments are required for the maintenance of myelinated peripheral nervous system axons.

2-DE and MALDI-TOF-MS for a comparative analysis of proteins expressed in different cellular models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disorder characterized by the selective loss of motor neurons from the spinal cord and brain. About 10% of ALS cases are familial (FALS), and in 20% of these cases the disease has been linked to mutations in the Cu,Zn-SOD1 gene. Although the molecular mechanisms causing these forms of ALS are still unclear, evidence has been provided that motor neurons injuries associated with mutant superoxide dismutase (SOD1)-related FALS result from a toxic gain-in-fuction of the mutated enzyme. To understand better the role of these mutations in the pathophysiology of FALS we have compared the pattern of proteins expressed in human neuroblastoma SH-SY5Y cell line with those of cell lines transfected with plasmids expressing the wild-type human SOD1 and the H46R and G93A mutants. 2-DE coupled to MALDI-TOF-MS were the proteomic tools used for identification of differentially expressed proteins. These included cytoskeletal proteins, proteins that regulate energetic metabolism and intracellular redox conditions, and the ubiquitin proteasome system. The proteomic approach allowed to expand the knowledge on the pattern of proteins, with altered expression, which we should focus on, for a better understanding of the possible mechanism involved in mutated-SOD1 toxicity. The cellular models considered in this work have also evidenced biochemical characteristics common to other SOD1-mutated cellular lines connected to the pathogenesis of ALS.

Neurofilament-M interacts with the D1 dopamine receptor to regulate cell surface expression and desensitization.

We used the yeast two-hybrid assay to identify novel proteins that interact with the D(1) dopamine receptor. The third cytoplasmic loop (residues 217-273) of the rat D(1) receptor was used as bait to identify clones encoding interacting proteins from a rat brain cDNA library. This identified two clones encoding the C terminus of rat neurofilament-M (NF-M) (residues 782-846). The NF-M clone did not interact with the third cytoplasmic loops of the rat D(2), D(3), or D(4) receptors, but showed weak interaction with that of the D(5) receptor. Coexpression of full-length NF-M with the D(1) receptor in HEK-293 cells resulted in >50% reduction of receptor binding accompanied by a reduction in D(1) receptor-mediated cAMP accumulation. NF-M had no effect on the expression of other dopamine receptor subtypes. Using a D(1) receptor-green fluorescent protein chimera and confocal fluorescence microscopy, we found that NF-M reduced D(1) receptor expression at the cell surface and promoted accumulation of the receptor in the cytosol. Interestingly, the D(1) receptors that were expressed at the cell surface in the presence of NF-M were resistant to agonist-induced desensitization. Cellular colocalization of NF-M and the D(1) receptor in the rat brain was examined by epifluorescence microscopy. These experiments showed that approximately 50% of medium-sized striatal neurons expressed both proteins. Colocalization was also observed in pyramidal cells and interneurons within the frontal cortex. Similar immunohistochemical analyses using NF-M-deficient mice showed decrements in D(1) receptor expression compared with control mice. These results suggest that NF-M interacts with the D(1) receptor in vivo and may modify its expression and regulation.

Changes of GTP binding proteins, not neurofilament-associated proteins, in the brain of the neurofilament-deficient quail, "Quiver".

A neurofilament (NF)-deficient mutant of the Japanese quail was named "Quiver", as it showed generalized quivering as a clinical sign. NF consists of three major subunits; low, middle and high. We previously reported that the noradrenaline and 5-hydroxytryptamine content in the neostriatum of the Quiver's brain was different from that in the normal quail, although disappearance of the three NF proteins occurred in all areas of the Quiver's brain. Thus, NF-related proteins may show considerable changes in the specific sites of Quiver's brain. In this study, an examination was made of the changes in NF-related proteins in the Quiver, by immunoblotting analysis. The amounts of cyclin-dependent kinase 5 (cdk5), which phosphorylates NF proteins, and tau which is a substrate of cdk5, in the neostriatum of the Quiver, were essentially the same as those in the normal quail, although NF proteins could not be detected in the Quiver. The amount of alpha-tubulin in the Quiver's brain was similar to that in the normal quail. Next, we investigated the changes of GTP binding (G) proteins in the Quiver's brain, because cytoskeletal components such as tubulin and F-actin bind with G proteins. [32P]ADP-ribosylation of G proteins (Gs by cholera toxin and Gi/G0 by pertussis toxin) in the neostriatum of the Quiver increased significantly, although alpha subunits of G proteins showed no change by immunoblotting analysis. The ratios of the trimer form in G proteins thus appear to increase more in the NF-deficient Quiver brain than in the brain of the normal quail. The G proteins-mediated adenylate cyclase activities were the same in the brain of both the Quiver and the normal quail. Possible interactions between NFs and G proteins are discussed.