Don W. Cleveland

An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria

Mutations in Cu/Zn superoxide dismutase (SOD1) cause a subset of cases of familial amyotrophic lateral sclerosis. Four lines of mice accumulating one of these mutant proteins (G37R) develop severe, progressive motor neuron disease. At lower levels of mutant accumulation, pathology is restricted to lower motor neurons, whereas higher levels cause more severe abnormalities and affect a variety of other neuronal populations. The most obvious cellular abnormality is the presence in axons and dendrites of membrane-bounded vacuoles, which appear to be derived from degenerating mitochondria. Since multiple lines of mice expressing wild-type human SOD1 at similar and higher levels do not show disease, the disease in mice expressing the G37R mutant SOD1 must arise from the acquisition of an adverse property by the mutant enzyme, rather than elevation or loss of SOD1 activity.

Number and evolutionary conservation of α- and β-tubulin and cytoplasmic β- and γ-actin genes using specific cloned cDNA probes

Abstract Bacterial clones containing inserted DNA sequences specific for α-tubulin, β-tubulin, β-actin and γ-actin have been constructed from mRNA of embryonic chick brain. Plasmids containing approximately 75, 90 and >90%, respectively, of the sequences present in α-tubulin, β-tubulin and β-actin mRNAs have been isolated as well as clones containing parts of the extensive 3′ untranslated regions of the β- and γ-actin mRNAs. The sequences for the two tubulins do not cross hybridize. Hybridization of labeled, cloned probes for each of the tubulins with chicken DNA digested with several restriction endonucleases reveals about four fragments for α- and four for β-tubulin. This seems to be the number of genes, since both the 5′ and 3′ ends of either cloned tubulin cDNAs hybridize to at least four common fragments in genomic DNA which has been digested with restriction endonucleases. The tubulin probes are able to hybridize under stringent conditions to DNA of all vertebrate genomes tested, as well as to sea urchin DNA, but not to yeast DNA. In digested sea urchin sperm DNA there are more than 20 different fragments which hybridize to both the 5′ and 3′ ends of the tubulin cDNAs. A full-length β-actin cDNA clone hybridizes to 4–7 bands in restricted chicken DNA and cross hybridizes to DNA from every other species tested, including sea urchin and yeast. Hybridization to chicken DNA of cloned probes specific for the 3′ untranslated regions of β- and γ-actin mRNA indicates that the β sequence is present only once in the genome and the γ is present in at most three copies. Neither 3′ untranslated sequence is conserved evolutionarily.

Onset and progression in inherited ALS determined by motor neurons and microglia

Dominant mutations in superoxide dismutase cause amyotrophic lateral sclerosis (ALS), a progressive paralytic disease characterized by loss of motor neurons. With the use of mice carrying a deletable mutant gene, expression within motor neurons was shown to be a primary determinant of disease onset and of an early phase of disease progression. Diminishing the mutant levels in microglia had little effect on the early disease phase but sharply slowed later disease progression. Onset and progression thus represent distinct disease phases defined by mutant action within different cell types to generate non–cell-autonomous killing of motor neurons; these findings validate therapies, including cell replacement, targeted to the non-neuronal cells.

From Charcot to Lou Gehrig: Deciphering selective motor neuron death in ALS

Since its description by Charcot more than 130 years ago, the mechanism underlying the characteristic selective degeneration and death of motor neurons in amyotrophic lateral sclerosis has remained a mystery. Modern genetics has now identified mutations in two genes — SOD1 and ALS2 — as primary causes of the disease, and has implicated others as potential contributors. Insights into these abnormalities, together with errors in the handling of synaptic glutamate and the potential excitotoxic response that this alteration provokes, have provided leads for the development of new strategies to identify an as yet elusive remedy for this progressive, fatal disorder.

ALS-Linked SOD1 Mutant G85R Mediates Damage to Astrocytes and Promotes Rapidly Progressive Disease with SOD1-Containing Inclusions

High levels of familial Amyotrophic Lateral Sclerosis (ALS)-linked SOD1 mutants G93A and G37R were previously shown to mediate disease in mice through an acquired toxic property. We report here that even low levels of another mutant, G85R, cause motor neuron disease characterized by an extremely rapid clinical progression, without changes in SOD1 activity. Initial indicators of disease are astrocytic inclusions that stain intensely with SOD1 antibodies and ubiquitin and SOD1-containing aggregates in motor neurons, features common with some cases of SOD1 mutant-mediated ALS. Astrocytic inclusions escalate markedly as disease progresses, concomitant with a decrease in the glial glutamate transporter (GLT-1). Thus, the G85R SOD1 mutant mediates direct damage to astrocytes, which may promote the nearly synchronous degeneration of motor neurons.

ALS: a disease of motor neurons and their nonneuronal neighbors.

Amyotrophic lateral sclerosis is a late-onset progressive neurodegenerative disease affecting motor neurons. The etiology of most ALS cases remains unknown, but 2% of instances are due to mutations in Cu/Zn superoxide dismutase (SOD1). Since sporadic and familial ALS affects the same neurons with similar pathology, it is hoped that therapies effective in mutant SOD1 models will translate to sporadic ALS. Mutant SOD1 induces non-cell-autonomous motor neuron killing by an unknown gain of toxicity. Selective vulnerability of motor neurons likely arises from a combination of several mechanisms, including protein misfolding, mitochondrial dysfunction, oxidative damage, defective axonal transport, excitotoxicity, insufficient growth factor signaling, and inflammation. Damage within motor neurons is enhanced by damage incurred by nonneuronal neighboring cells, via an inflammatory response that accelerates disease progression. These findings validate therapeutic approaches aimed at nonneuronal cells.

On the road to cancer: Aneuploidy and the mitotic checkpoint

Abnormal chromosome content — also known as aneuploidy — is the most common characteristic of human solid tumours. It has therefore been proposed that aneuploidy contributes to, or even drives, tumour development. The mitotic checkpoint guards against chromosome mis-segregation by delaying cell-cycle progression through mitosis until all chromosomes have successfully made spindle-microtubule attachments. Defects in the mitotic checkpoint generate aneuploidy and might facilitate tumorigenesis, but more severe disabling of checkpoint signalling is a possible anticancer strategy.

Centromeres and Kinetochores: From Epigenetics to Mitotic Checkpoint Signaling

The centromere is a chromosomal locus that ensures delivery of one copy of each chromosome to each daughter at cell division. Efforts to understand the nature and specification of the centromere have demonstrated that this central element for ensuring inheritance is itself epigenetically determined. The kinetochore, the protein complex assembled at each centromere, serves as the attachment site for spindle microtubules and the site at which motors generate forces to power chromosome movement. Unattached kinetochores are also the signal generators for the mitotic checkpoint, which arrests mitosis until all kinetochores have correctly attached to spindle microtubules, thereby representing the major cell cycle control mechanism protecting against loss of a chromosome (aneuploidy).

Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis

Dominant mutations in superoxide dismutase cause amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disease that is characterized by the loss of motor neurons. Using mice carrying a deletable mutant gene, diminished mutant expression in astrocytes did not affect onset, but delayed microglial activation and sharply slowed later disease progression. These findings demonstrate that mutant astrocytes are viable targets for therapies for slowing the progression of non–cell autonomous killing of motor neurons in ALS.

Focal loss of the glutamate transporter EAAT2 in a transgenic rat model of SOD1 mutant-mediated amyotrophic lateral sclerosis (ALS).

Transgenic overexpression of Cu+2/Zn+2 superoxide dismutase 1 (SOD1) harboring an amyotrophic lateral sclerosis (ALS)-linked familial genetic mutation (SOD1G93A) in a Sprague–Dawley rat results in ALS-like motor neuron disease. Motor neuron disease in these rats depended on high levels of mutant SOD1 expression, increasing from 8-fold over endogenous SOD1 in the spinal cord of young presymptomatic rats to 16-fold in end-stage animals. Disease onset in these rats was early, ≈115 days, and disease progression was very rapid thereafter with affected rats reaching end stage on average within 11 days. Pathological abnormalities included vacuoles initially in the lumbar spinal cord and subsequently in more cervical areas, along with inclusion bodies that stained for SOD1, Hsp70, neurofilaments, and ubiquitin. Vacuolization and gliosis were evident before clinical onset of disease and before motor neuron death in the spinal cord and brainstem. Focal loss of the EAAT2 glutamate transporter in the ventral horn of the spinal cord coincided with gliosis, but appeared before motor neuron/axon degeneration. At end-stage disease, gliosis increased and EAAT2 loss in the ventral horn exceeded 90%, suggesting a role for this protein in the events leading to cell death in ALS. These transgenic rats provide a valuable resource to pursue experimentation and therapeutic development, currently difficult or impossible to perform with existing ALS transgenic mice.