Elize D. Haasdijk

CuZn superoxide dismutase (SOD1) accumulates in vacuolated mitochondria in transgenic mice expressing amyotrophic lateral sclerosis-linked SOD1 mutations

Abstract Cytosolic Cu/Zn superoxide dismutase (SOD1) is a ubiquitous small cytosolic metalloenzyme, which catalyses the conversion of superoxide anion to hydrogen peroxide. Mutations in the SOD1 gene cause a familial form of amyotrophic lateral sclerosis (fALS). The mechanism by which mutant SOD1s cause the degeneration of motor neurons is not understood. Transgenic mice expressing multiple copies of fALS-mutant SOD1s develop an ALS-like motor neuron disease. Vacuolar degeneration of mitochondria has been identified as the main pathological feature associated with motor neuron death and paralysis in several lines of fALS-SOD1 mice. Using confocal and electron microscopy we show that mutant SOD1 is present at a high concentration in vacuolated mitochondria, where it colocalises with cytochrome c. Mutant SOD1 is also present in mildly swollen mitochondria prior to the appearance of vacuoles, suggesting that the leakage or translocation of mutant human SOD1 in mitochondria may be the primary event triggering their further degeneration. Vacuolated mitochondria containing SOD1 also occur in transgenic mice expressing a high concentration of wild-type human SOD1. In sum, our data suggest that both fALS-mutant and wild-type SOD1 may cross the mitochondrial outer membrane, and by doing so induce the degeneration of these mitochondria.

Neuron-Specific Expression of Mutant Superoxide Dismutase Is Sufficient to Induce Amyotrophic Lateral Sclerosis in Transgenic Mice

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), an adult-onset progressive paralytic disease characterized by loss of motor neurons, and cause an ALS-like disease when expressed in mice. Recent data have suggested that motor neuron degeneration results from toxic actions of mutant SOD1 operating in both motor neurons and their neighboring glia, raising the question whether mutant SOD1 expression selectively in neurons is sufficient to induce disease. Here we show that neuronal expression of mutant SOD1 is sufficient to cause motor neuron degeneration and paralysis in transgenic mice with cytosolic dendritic ubiquitinated SOD1 aggregates as the dominant pathological feature. In addition, we show that crossing our neuron-specific mutant SOD1 mice with ubiquitously wild-type SOD1-expressing mice leads to dramatic wild-type SOD1 aggregation in oligodendroglia after the onset of neuronal degeneration. Together, our findings support a pathogenic scenario in which mutant SOD1 in neurons triggers neuronal degeneration, which in turn may facilitate aggregate formation in surrounding glial cells.

Bicaudal D2, Dynein, and Kinesin-1 Associate with Nuclear Pore Complexes and Regulate Centrosome and Nuclear Positioning during Mitotic Entry

Mammalian Bicaudal D2 is the missing molecular link between cytoplasmic motor proteins and the nucleus during nuclear positioning prior to the onset of mitosis.

Motor Neuron Disease-Associated Mutant Vesicle-Associated Membrane Protein-Associated Protein (VAP) B Recruits Wild-Type VAPs into Endoplasmic Reticulum-Derived Tubular Aggregates

The vesicle-associated membrane protein-associated proteins (VAPs) VAPA and VAPB interact with lipid-binding proteins carrying a short motif containing two phenylalanines in an acidic tract (FFAT motif) and targets them to the cytosolic surface of the endoplasmic reticulum (ER). A genetic mutation (P56S) in the conserved major sperm protein homology domain of VAPB has been linked to motor-neuron degeneration in affected amyotrophic lateral sclerosis (ALS) patients. We report that in the CNS, VAPB is abundant in motor neurons and that the P56S substitution causes aggregation of mutant VAPB in immobile tubular ER clusters, perturbs FFAT-motif binding, and traps endogenous VAP in mutant aggregates. Expression of mutant VAPB or reduction of VAP by short hairpin RNA in primary neurons causes Golgi dispersion and cell death. VAPA and VAPB are reduced in human ALS patients and superoxide dismutase 1 (SOD1)-ALS-transgenic mice, suggesting that VAP family proteins may be involved in the pathogenesis of sporadic and SOD1-linked ALS. Our data support a model in which reduced levels of VAP family proteins result in decreased ER anchoring of lipid-binding proteins and cause motor neuron degeneration.

Reduced levels of cholesterol, phospholipids, and fatty acids in cerebrospinal fluid of Alzheimer disease patients are not related to apolipoprotein E4

Apolipoprotein E4 (apoE4) has been identified as a major risk factor for Alzheimer disease (AD). Previously it has been reported that levels of apoE are reduced in cerebrospinal fluid (CSF) of AD patients. Because it is known that apoE4 affects plasma lipid metabolism, we examined whether the presence of apoE4 might correlate with an altered lipid metabolism in the CSF of control subjects and AD patients. ApoE and lipid concentrations were determined in postmortem ventricular CSF of 30 neuropathologically confirmed AD cases and 31 age-matched control patients. ApoE genotyping was performed on frozen brain tissue of the same patients. In line with other reports, we found an increased APOE*4 allele frequency in the AD group (0.461) when compared with the control group (0.225). ApoE levels in CSF of AD patients were not significantly reduced when compared with the controls (mean ± SD: 63 ± 55 and 82 ± 62 μg/dL for AD and controls, respectively). However, in the CSF of AD patients levels of free and esterified cholesterol (0.13 ± 0.09 and 0.25 ± 0.19 mg/dL, and 0.25 ± 0.19 and 0.42 ± 0.34, respectively), phospholipids (0.2 ± 0.1 and 3.5 ± 5.0 mg/dL) and, suprisingly, also fatty acids (4.5 ± 3.2 and 28.0 ± 18.5 μmol/L) were found to be significantly reduced. After correction for age, sex, postmortem delay, and pH the levels of phospholipids, fatty acids, and free cholesterol were still significantly reduced (p = 0.021, p = 0.026, and p = 0.012, respectively). The apoE and lipid levels in CSF of AD-and control patients appeared not to be affected by the number of APOE*4 alleles. In conclusion, our results suggest an altered lipid homeostasis in the brain of AD patients that is not related to the presence of apoE4. It is, therefore, unlikely that an effect of apoE4 on brain lipid metabolism is the underlying mechanism behind the role of apoE4 in the development of AD.

ATF3 expression precedes death of spinal motoneurons in amyotrophic lateral sclerosis-SOD1 transgenic mice and correlates with c-Jun phosphorylation, CHOP expression, somato-dendritic ubiquitination and Golgi fragmentation

To obtain insight into the morphological and molecular correlates of motoneuron degeneration in amyotrophic lateral sclerosis (ALS) mice that express G93A mutant superoxide dismutase (SOD)1 (G93A mice), we have mapped and characterized ‘sick’ motoneurons labelled by the ‘stress transcription factors’ ATF3 and phospho‐c‐Jun. Immunocytochemistry and in situ hybridization showed that a subset of motoneurons express ATF3 from a relatively early phase of disease before the onset of active caspase 3 expression and motoneuron loss. The highest number of ATF3‐expressing motoneurons occurred at symptom onset. The onset of ATF3 expression correlated with the appearance of ubiquitinated neurites. Confocal double‐labelling immunofluorescence showed that all ATF3‐positive motoneurons were immunoreactive for phosphorylated c‐Jun. Furthermore, the majority of ATF3 and phospho‐c‐Jun‐positive motoneurons were also immunoreactive for CHOP (GADD153) and showed Golgi fragmentation. A subset of ATF3 and phosphorylated c‐Jun‐immunoreactive motoneurons showed an abnormal appearance characterized by a number of distinctive features, including an eccentric flattened nucleus, perikaryal accumulation of ubiquitin immunoreactivity, juxta‐nuclear accumulation of the Golgi apparatus and the endoplasmic reticulum, and intense Hsp70 immunoreactivity. These abnormal cells were not immunoreactive for active caspase 3. We conclude that motoneurons in ALS‐SOD1 mice prior to their death and disappearance experience a prolonged sick phase, characterized by the gradual accumulation of ubiquitinated material first in the neurites and subsequently the cell body.

Decrease of Hsp25 protein expression precedes degeneration of motoneurons in ALS‐SOD1 mice

We have investigated the expression of Hsp25, a heat shock protein constitutively expressed in motoneurons, in amyotrophic lateral sclerosis (ALS) mice that express G93A mutant SOD1 (G93A mice). Immunocytochemistry and Western blotting showed that a decrease of Hsp25 protein expression occurred in motoneurons of G93A mice prior to the onset of motoneuron death and muscle weakness. This decrease in Hsp25 expression also preceded the appearance of SOD1 aggregates as identified by cellulose acetate filtration and Western blot analysis. In contrast to Hsp25 protein levels, Hsp25 mRNA as determined by in situ hybridization and RT‐PCR, remained unchanged. This suggests that the decrease in Hsp25 protein levels occurs post‐transcriptionally. In view of the cytoprotective properties of Hsp25 and the temporal relationship between decreased Hsp25 expression and the onset of motoneuron death, it is feasible that reduced Hsp25 concentration contributes to the degeneration of motoneurons in G93A mice. These data are consistent with the idea that mutant SOD1 may reduce the availability of the protein quality control machinery in motoneurons.

Estradiol improves cerebellar memory formation by activating estrogen receptor β

Learning motor skills is critical for motor abilities such as driving a car or playing piano. The speed at which we learn those skills is subject to many factors. Yet, it is not known to what extent gonadal hormones can affect the achievement of accurate movements in time and space. Here we demonstrate via different lines of evidence that estradiol promotes plasticity in the cerebellar cortex underlying motor learning. First, we show that estradiol enhances induction of long-term potentiation at the parallel fiber to Purkinje cell synapse, whereas it does not affect long-term depression; second, we show that estradiol activation of estrogen receptor β receptors in Purkinje cells significantly improves gain-decrease adaptation of the vestibulo-ocular reflex, whereas it does not affect general eye movement performance; and third, we show that estradiol increases the density of parallel fiber to Purkinje cell synapses, whereas it does not affect the density of climbing fiber synapses. We conclude that estradiol can improve motor skills by potentiating cerebellar plasticity and synapse formation. These processes may be advantageous during periods of high estradiol levels of the estrous cycle or pregnancy.

Unmyelinated and myelinated skin nerve damage in Guillain–Barré syndrome: Correlation with pain and recovery

Summary Unmyelinated and myelinated skin nerves are diffusely affected in Guillain–Barré syndrome and its variants. Intraepidermal nerve fiber density declines early, remains low over time, correlates with pain severity in the acute phase, and may predict long‐term disability. Abstract We performed a prospective study in 32 patients with Guillain–Barré syndrome (GBS) or its variants to correlate intraepidermal nerve fiber density (IENFD) at the distal leg and lumbar region with pain, autonomic dysfunction, and outcome. In the acute phase, IENFD was reduced in 60% and 61.9% of patients at the distal leg and lumbar region, respectively. In the acute phase, 43.7% of patients complained of neuropathic pain. Their IENFD at the distal leg was significantly lower than in patients without pain (P < .001) and correlated with pain intensity (rs = −0.51; P = .003). Intriguingly, also patients with the pure motor variant of GBS and pain had low IENFD. At 6‐month follow‐up, only 3 patients complained of persisting neuropathic pain, whereas 3 patients reported late‐onset pain symptoms. IENFD in the acute phase did not predict presence or intensity of pain at 6‐month follow‐up. IENFD in the acute phase did not correlate with clinical dysautonomia or GBS severity at nadir. However, it correlated with poorer GBS disability score at 6 months (P = .04), GBS score at nadir (P = .03), and clinically probable dysautonomia (P = .004). At 6‐month follow‐up, median IENFD remained significantly low both at the distal leg (P = .024) and lumbar region (P = .005). Double and triple staining confocal microscope studies showed diffuse damage of myelinated dermal nerves along with axonal degeneration, and mononuclear cell infiltration. Unmyelinated and myelinated skin nerves are diffusely affected in GBS and its variants, including the pure motor form. IENFD declines early, remains low over time, correlates with pain severity in the acute phase, and may predict long‐term disability.

Spinal inhibitory interneuron pathology follows motor neuron degeneration independent of glial mutant superoxide dismutase 1 expression in SOD1-ALS mice.

Motor neuron degeneration and skeletal muscle denervation are hallmarks of amyotrophic lateral sclerosis (ALS), but other neuron populations and glial cells are also involved in ALS pathogenesis. We examined changes in inhibitory interneurons in spinal cords of the ALS model low-copy Gurney G93A-SOD1 (G1del) mice and found reduced expression of markers of glycinergic and GABAergic neurons, that is, glycine transporter 2 (GlyT2) and glutamic acid decarboxylase (GAD65/67), specifically in the ventral horns of clinically affected mice. There was also loss of GlyT2 and GAD67 messenger RNA-labeled neurons in the intermediate zone. Ubiquitinated inclusions appeared in interneurons before 20 weeks of age, that is, after their development in motor neurons but before the onset of clinicalsigns and major motor neuron degeneration, which starts from 25weeks of age. Because mutant superoxide dismutase 1 (SOD1) in glia might contribute to the pathogenesis, we also examined neuron-specific G93A-SOD1 mice; they also had loss of inhibitory interneuron markers in ventral horns and ubiquitinated interneuron inclusions. These data suggest that, in mutant SOD1-associated ALS, pathological changes may spread from motor neurons to interneuronsin a relatively early phase of the disease, independent of the presence of mutant SOD1 in glia. The degeneration of spinal inhibitory interneurons may in turn facilitate degeneration of motor neurons and contribute to disease progression.