Raymond P. Roos

Linkage of a gene causing familial amyotrophic lateral sclerosis to chromosome 21 and evidence of genetic-locus heterogeneity

BACKGROUND Amyotrophic lateral sclerosis is a progressive neurologic disorder that commonly results in paralysis and death. Despite more than a century of research, no cause of, cure for, or means of preventing this disorder has been found. In a minority of cases, it is familial and inherited as an autosomal dominant trait with age-dependent penetrance. In contrast to the sporadic form of amyotrophic lateral sclerosis, the familial form provides the opportunity to use molecular genetic techniques to localize an inherited defect. Furthermore, such studies have the potential to discover the basic molecular defect causing motor-neuron degeneration. METHODS AND RESULTS We evaluated 23 families with familial amyotrophic lateral sclerosis for linkage of the gene causing this disease to four DNA markers on the long arm of chromosome 21. Multipoint linkage analyses demonstrated linkage between the gene and these markers. The maximum lod score--5.03--was obtained 10 centimorgans distal (telomeric) to the DNA marker D21S58. There was a significant probability (P less than 0.0001) of genetic-locus heterogeneity in the families. CONCLUSIONS The localization of a gene causing familial amyotrophic lateral sclerosis provides a means of isolating this gene and studying its function. Insight gained from understanding the function of this gene may be applicable to the design of rational therapy for both the familial and sporadic forms of the disease.

Late Denervation in Patients with Antecedent Paralytic Poliomyelitis

The development of new weakness, fatigue, and pain decades after acute paralytic poliomyelitis is a recognized syndrome. We conducted a controlled study of this syndrome by analyzing clinical, electromyographic, and muscle-biopsy features in 18 patients with a history of poliomyelitis--13 reporting 1 to 20 years of new weakness and 5 without new symptoms. The patients with new weakness also reported new muscle atrophy (9 of 13) and fatigue (10 of 13), symptoms not reported by the controls. The age at the time of acute poliomyelitis, severity of poliomyelitis, residual disability, number of years since acute poliomyelitis, and age at the time of study were comparable in the weakening and control groups. Evidence of remote denervation consistent with antecedent poliomyelitis was demonstrated in all patients by electromyography or muscle biopsy or both. In addition, active denervation (as evidenced by spontaneous activity on conventional electromyography, increased jitter on single-fiber electromyography, or atrophic myofibers) was found in 12 patients in the weakening group and in all 5 controls. Immunohistochemical detection of myofibers expressing the neural-cell adhesion molecule corroborated ongoing denervation in both patient groups. When muscle data from both groups were pooled, correlations were observed between the extent of past reinnervation and the degree of ongoing motor-unit instability. We conclude that the extensive reinnervation of denervated muscle that occurs in paralytic poliomyelitis may be followed by late denervation of the previously reinnervated muscle fibers. Electromyographic and muscle-biopsy evidence of ongoing denervation does not distinguish between stable patients with prior paralytic poliomyelitis and those with new weakness.

Ca2+ and reactive oxygen species in staurosporine-induced neuronal apoptosis

Abstract: Staurosporine (0.03–0.5 µM) induced a dose‐dependent, apoptotic degeneration in cultured rat hippocampal neurons that was sensitive to 24‐h pretreatments with the protein synthesis inhibitor cycloheximide (1 µM) or the cell cycle inhibitor mimosine (100 µM). To investigate the role of Ca2+ and reactive oxygen species in staurosporine‐induced neuronal apoptosis, we overexpressed calbindin D28K, a Ca2+ binding protein, and Cu/Zn superoxide dismutase, an antioxidative enzyme, in the hippocampal neurons using adenovirus‐mediated gene transfer. Infection of the cultures with the recombinant adenoviruses (100 multiplicity of infection) resulted in a stable expression of the respective proteins assessed 48 h later. Overexpression of both calbindin D28K and Cu/Zn superoxide dismutase significantly reduced staurosporine neurotoxicity compared with control cultures infected with a β‐galactosidase overexpressing adenovirus. Staurosporine‐induced neuronal apoptosis was also significantly reduced when the culture medium was supplemented with 10 or 30 mM K+, suggesting that Ca2+ influx via voltage‐sensitive Ca2+ channels reduces this apoptotic cell death. In contrast, neither the glutamate receptor agonist NMDA (1–10 µM) nor the NMDA receptor antagonist dizocilpine (MK‐801; 1 µM) was able to reduce staurosporine neurotoxicity. Cultures treated with the antioxidants U‐74500A (1–10 µM) and N‐acetylcysteine (100 µM) also demonstrated reduced staurosporine neurotoxicity. These results suggest a fundamental role for both Ca2+ and reactive oxygen species in staurosporine‐induced neuronal apoptosis.

Astrocyte loss of mutant SOD1 delays ALS disease onset and progression in G85R transgenic mice

Approximately 10% of patients with amyotrophic lateral sclerosis (ALS) have familial ALS (FALS), and 20% of FALS are caused by mutations of superoxide dismutase type 1 (MTSOD1). The fact that some MTSOD1s that cause FALS have full dismutase activity (e.g. G37R) and others no dismutase activity (e.g. G85R) suggests that MTSOD1 causes FALS due to toxicity of the protein rather than a loss in enzymatic function. Compelling data have demonstrated that motor neuron (MN) degeneration can result from a non-cell autonomous effect of the MTSOD1. In order to clarify the role of astrocytes in FALS, we deleted MTSOD1 in astrocytes of G85R transgenic mice. In contrast to a similar study using G37R mice in which astrocyte MTSOD1 loss affected only the late phase of ALS disease, we found that astrocyte MTSOD1 loss in G85R mice delayed disease onset and prolonged the early phase of disease progression, without affecting the late phase. In addition, astrocyte G85R knockdown resulted in decreased microgliosis, decreased SOD1-immunoreactive inclusions and preservation of GLT-1 transporter expression. The differential effects of astrocyte G85R versus G37R knockdown on MN death demonstrate SOD1 mutation-specific effects on ALS pathogenesis; these differences may be a result of the different dismutase activities of the two mutants. The effect of the knockdown of G85R expression in astrocytes on onset as well as disease duration highlights the importance of this cell type in FALS.

The Unfolded Protein Response in Familial Amyotrophic Lateral Sclerosis

Mutant superoxide dismutase type 1 (MTSOD1) is thought to cause ∼20% of cases of familial amyotrophic lateral sclerosis (FALS) because it misfolds and aggregates. Previous studies have shown that MTSOD1 accumulates inside the endoplasmic reticulum (ER) and activates the unfolded protein response (UPR), suggesting that ER stress is involved in the pathogenesis of FALS. We used a genetic approach to investigate the role of the UPR in FALS. We crossed G85RSOD1 transgenic mice with pancreatic ER kinase haploinsufficient (PERK(+/-)) mice to obtain G85R/PERK(+/-) mice. PERK(+/-) mice carry a loss of function mutation of PERK, which is the most rapidly activated UPR pathway, but have no abnormal phenotype. Compared with G85R transgenic mice, G85R/PERK(+/-) mice had a dramatically accelerated disease onset as well as shortened disease duration and lifespan. There was also acceleration of the pathology and earlier MTSOD1 aggregation. A diminished PERK response accelerated disease and pathology in G85R transgenic mice presumably because the mice had a reduced capacity to turn down synthesis of misfolded SOD1, leading to an early overloading of the UPR. The results indicate that the UPR has a significant influence on FALS, and suggest that enhancing the UPR may be effective in treating ALS.

Glutamate carboxypeptidase II inhibition protects motor neurons from death in familial amyotrophic lateral sclerosis models

Approximately 10% of cases of amyotrophic lateral sclerosis (ALS), a progressive and fatal degeneration that targets motor neurons (MNs), are inherited, and ≈20% of these cases of familial ALS (FALS) are caused by mutations of copper/zinc superoxide dismutase type 1. Glutamate excitotoxicity has been implicated as a mechanism of MN death in both ALS and FALS. In this study, we tested whether a neuroprotective strategy involving potent and selective inhibitors of glutamate carboxypeptidase II (GCPII), which converts the abundant neuropeptide N-acetylaspartylglutamate to glutamate, could protect MNs in an in vitro and animal model of FALS. Data suggest that the GCPII inhibitors prevented MN cell death in both of these systems because of the resultant decrease in glutamate levels. GCPII inhibition may represent a new therapeutic target for the treatment of ALS.

The effect of mutant SOD1 dismutase activity on non-cell autonomous degeneration in familial amyotrophic lateral sclerosis

Mutant superoxide dismutase type 1 (MTSOD1), the most common known cause of familial amyotrophic lateral sclerosis (FALS), is believed to cause FALS as a result of a toxicity of the protein. MTSOD1s with full dismutase enzymatic activity (e.g., G37R) and without any enzymatic activity (e.g., G85R) cause FALS, demonstrating that the ability of MTSOD1 to cause FALS is not dependent on the dismutase activity; however, it remains unclear whether MTSOD1 dismutase activity can influence disease phenotype. In the present study, we selectively knocked down G85R expression in particular cell types of G85R mice. Results following knockdown of G85R in motor neurons (MNs)/interneurons of G85R mice were similar to results from a published study involving knockdown of G37R in G37R mice; however, G85R knockdown in microglia/macrophages induced a prolonged early and late disease phase while G37R knockdown in the same cells only affected late phase. These results show that: (i) MN as well as non-MN expression of G85R, like G37R, has a significant effect on disease in transgenic mice - indicating the role of non-cell autonomous degeneration in both dismutase-active and inactive MTSOD1s. (ii) The effect of MTSOD1 expression in microglia/macrophages varies with different mutants, and may be influenced by the MTSOD1s dismutase activity.

Cell-specific proteins regulate viral RNA translation and virus-induced disease

Translation initiation of the picornavirus genome is regulated by an internal ribosome entry site (IRES). The IRES of a neurovirulent picornavirus, the GDVII strain of Theilers murine encephalomyelitis virus, requires polypyrimidine tract‐binding protein (PTB) for its function. Although neural cells are deficient in PTB, they express a neural‐specific homologue of PTB (nPTB). We now show that nPTB and PTB bind similarly to multiple sites in the GDVII IRES, rendering it competent for efficient translation initiation. Mutation of a PTB or nPTB site results in a more prominent decrease in nPTB than PTB binding, a decrease in activity of nPTB compared with PTB in promoting translation initiation, and attenuation of the neurovirulence of the virus without a marked effect on virus growth in non‐neural cells. The addition of a second‐site mutation in the mutant IRES generates a new PTB (nPTB) binding site, and restores nPTB binding, translation initiation and neurovirulence. We conclude that the tissue‐specific expression and differential RNA‐binding properties of PTB and nPTB are important determinants of cell‐specific translational control and viral neurovirulence.

Wild-type SOD1 overexpression accelerates disease onset of a G85R SOD1 mouse

Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (FALS), and approximately 25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase type 1 (SOD1). Mutant (MT) SOD1 is thought to be pathogenic because it misfolds and aggregates. A number of transgenic mice have been generated that express different MTSOD1s as transgenes and exhibit an ALS-like disease. Although one study found that overexpression of human wild-type (WT) SOD1 did not affect disease in G85R transgenic mice, more recent reports claim that overexpression of WTSOD1 in other MTSOD1 transgenic mice hastened disease, raising a possibility that the effect of WTSOD1 overexpression in this FALS mouse model is mutant-specific. In order to clarify this issue, we studied the effect of WTSOD1 overexpression in a G85R transgenic mouse that we recently generated. We found that G85R/WTSOD1 double transgenic mice had an acceleration of disease onset and shortened survival compared with G85R single transgenic mice; in addition, there was an earlier appearance of pathological and immunohistochemical abnormalities. The spinal cord insoluble fraction from G85R/WTSOD1 mice had evidence of G85R-WTSOD1 heterodimers and WTSOD1 homodimers (in addition to G85R homodimers) with intermolecular disulfide bond cross-linking. These studies suggest that WTSOD1 can be recruited into disease-associated aggregates by redox processes, providing an explanation for the accelerated disease seen in G85R mice following WTSOD1 overexpression, and suggesting the importance of incorrect disulfide-linked protein as key to MTSOD1 toxicity.

Detection of visna virus antigens and RNA in glial cells in foci of demyefnation

Visna is a slow virus infection of sheep in which the characteristic pathological change is demyelination in foci of inflammation. The latter is thought to be the result of an immunopathological process directed against cellular and antigenic targets that have been difficult to define because of restricted viral gene expression. A new simultaneous detection assay is used to demonstrate viral RNA in cells identified unambiguously as oligodendrocytes and astrocytes. These cells were found in inflammatory foci. With a new strain of virus that causes a rapid form of visna in Icelandic sheep, viral antigens were demonstrated in cells in the inflammatory lesions. These findings are consistent with the postulated immunopathological mechanism of demyelination: cells that maintain intact myelin sheaths in the central nervous system are destroyed by the inflammatory response to viral antigens expressed in these cells.