Peter C. Blumbergs

Stalning af amyloid percursor protein to study axonal damage in mild head Injury

The most common definition of cerebral concussion is that of a transient loss of neurological function without macroscopic or microscopic abnormality in the brain. However, some patients have persistent symptoms and subtle neuropsychological deficits, particularly affecting memory. We have studied five patients aged 59-89 years who sustained mild concussive head injury and died of other causes (2-99 days post-injury). Immunostaining with an antibody to amyloid precursor protein, a marker of fast axonal transport, showed multifocal axonal injury in all five. All had axonal damage in the fornices, which are important in memory function.

Rhabdomyolysis: A review

Rhabdomyolysis, a syndrome of skeletal muscle breakdown with leakage of muscle contents, is frequently accompanied by myoglobinuria, and if sufficiently severe, acute renal failure with potentially life‐threatening metabolic derangements may ensue. A diverse spectrum of inherited and acquired disorders affecting muscle membranes, membrane ion channels, and muscle energy supply causes rhabdomyolysis. Common final pathophysiological mechanisms among these causes of rhabdomyolysis include an uncontrolled rise in free intracellular calcium and activation of calcium‐dependent proteases, which lead to destruction of myofibrils and lysosomal digestion of muscle fiber contents. Recent advances in molecular genetics and muscle enzyme histochemistry may enable a specific metabolic diagnosis in many patients with idiopathic recurrent rhabdomyolysis.

Loss of brainstem serotonin- and substance P-containing neurons in Parkinson's disease.

Using postmortem immunohistochemical analysis, we have identified degeneration of several different neuronal cell groups in the brainstem of patients dying with idiopathic Parkinsons disease. We report the first chemically identified loss of presumed serotonin neurons in the median raphe nucleus of the pons and of substance P-containing preganglionic neurons in the dorsal motor vagal nucleus. This evidence is concordant with other evidence that the primary neuropathological process is not confined either to a single pathway or to neurons containing a particular transmitter. Rather it appears that Parkinsons disease affects several classes of neurons in localized areas of the brainstem.

In situ and in vitro study of colocalization and segregation of α-synuclein, ubiquitin, and lipids in Lewy bodies

alpha-Synuclein and ubiquitin are two Lewy body protein components that may play antagonistic roles in the pathogenesis of Lewy bodies. We examined the relationship between alpha-synuclein, ubiquitin, and lipids in Lewy bodies of fixed brain sections or isolated from cortical tissues of dementia with Lewy bodies. Lewy bodies exhibited a range of labeling patterns for alpha-synuclein and ubiquitin, from a homogeneous pattern in which alpha-synuclein and ubiquitin were evenly distributed and overlapped across the inclusion body to a concentric pattern in which alpha-synuclein and ubiquitin were partially segregated, with alpha-synuclein labeling concentrated in the peripheral domain and ubiquitin in the central domain of the Lewy body. Lipids represented a significant component in both homogeneous and concentric Lewy bodies. These results suggest that Lewy bodies are heterogeneous in their subregional composition. The segregation of alpha-synuclein to Lewy body peripheral domain is consistent with the hypothesis that alpha-synuclein is continually deposited onto Lewy bodies.

Multiple-system atrophy: a new α-synuclein disease?

Multiple-system atrophy is a neurodegenerative disease that involves various combinations of parkinsonism, ataxia, corticospinal motor signs, and postural hypotension or urinary incontinence (Shy-Drager syndrome). The pathological hallmark of the disease is the presence of glial and neuronal cytoplasmic inclusions, shown by modified Bielschowsky silver impregnation and, to a limited extent, with antisera to ubiquitin or B-crystallin. We show that glial cytoplasmic inclusions and degenerating neurites in multiple-system atrophy are strongly immunopositive for -synuclein, a presynaptic protein present in brainstem and cortical Lewy bodies and related neurites in idiopathic Parkinson’s disease and dementia with Lewy bodies, and, in a mutated form, in some cases of familial Parkinson’s disease. Antiserum against human -synuclein (residues 111–131) was raised in a rabbit. Paraffin-embedded sections (5 m) and frozen sections (50 m) from different brain regions were immunostained with the -synuclein antibody (1 in 1000–2000 dilution) and the avidin-biotin-peroxidase procedure. Immunostaining was not seen with preadsorbed or preimmune serum or in the absence of the primary antiserum. Brains from people with multiple-system atrophy (n=8) were obtained from the South Australian Brain Bank. The clinical diagnoses before death had included Parkinson’s disease, multiple-system atrophy with olivopontocerebellar involvement, and multiple-system atrophy with autonomic symptoms. The neuropathological diagnosis of multiplesystem atrophy was made by the demonstration of glial cytoplasmic inclusions with the modified Bielschowsky silver stain and immunostaining for ubiquitin and B-crystallin. We assessed the same regions in 12 brains judged to be normal, five from people who had had motor-neuron disease, and ten from people with idiopathic Parkinson’s disease, all also from the brain bank. Sections from brainstem, basal ganglia, cerebellar and cerebral cortices in the multiple-system-atrophy brains all showed strong immunostaining of glial cytoplasmic inclusions, mainly in white matter (figure). Pronounced -synuclein immunoreactivity was also seen in degenerating neurites (figure) and in the cytoplasmic and intranuclear inclusions of neurons in the putamen and pontine nuclei. In normal brains and those from people with motor-neuron disease, no abnormal -synuclein-containing structures were seen; immunostaining for -synuclein was limited to the normal background pattern, which shows the presence of the protein in nerve terminals. In idiopathic Parkinson’s disease, Lewy bodies and degenerating neurites were positive for synuclein, as shown previously, but no immunopositive glial cytoplasmic inclusions were seen. We used confocal microscopy and double-staining for synuclein (1 in 500) and ubiquitin (monoclonal antibody 1510, Chemicon, 1 in 200) to assess the colocalisation of these two markers in glial cytoplasmic inclusions and degenerating neurites in multiple-system atrophy. Small numbers of synuclein-positive degenerating neurites and inclusions were ubiquitin-positive, but -synuclein immunoreactivity was clearly more sensitive than ubiquitin immunoreactivity as a neuropathological marker for the lesions of multiple-system atrophy (figure). The pronounced increase in the number of multiplesystem-atrophy inclusions and neurites shown with the synuclein antibody (compared with the ubiquitin antibody) contrasts with the number in Parkinson’s disease and dementia with Lewy bodies, disorders in which the two antibodies show similar neuropathological abnormalities. Immunoreactivity for -synuclein occurs in plaque-associated neurites in Alzheimer’s disease, but is absent from neuronal and glial inclusions in Alzheimer’s disease and other neurodegenerative disorders. Antibodies to -synuclein

FUS mutations in amyotrophic lateral sclerosis: clinical, pathological, neurophysiological and genetic analysis

Objective FUS gene mutations were recently identified in familial amyotrophic lateral sclerosis (ALS). The present studies sought to define the clinical, post-mortem and neurophysiological phenotypes in ALS families with FUS mutations and to determine the frequency of FUS mutations in familial and sporadic ALS. Methods FUS was screened for mutations in familial and sporadic ALS cases. Clinical, post-mortem and neurophysiological features of large families with FUS mutations are described. Results and conclusions FUS mutations were evident in 3.2% (4/124) of familial ALS, representing the second most common gene abnormality to be described in familial ALS after SOD1. No mutations were present in 247 sporadic ALS cases. The clinical presentation in 49 affected patients was consistent with a predominantly lower motor neuron disorder, supported by post-mortem findings. Upper motor neuron involvement varied, with Wallerian degeneration of corticospinal tracts present in one post-mortem case but absent in a second case from the same family. Features of cortical hyperexcitability demonstrated upper motor neuron involvement consistent with other forms of familial and sporadic ALS. One case presented with frontotemporal dementia (FTD) indicating that this may be a rare presenting feature in families with FUS mutation. Ubiquitin-positive cytoplasmic skein-like inclusions were present in lower motor neurons, but in contrast to sporadic ALS, no TDP-43 pathology was evident. Mutation-specific clinical features were identified. Patients with a R521C mutation were significantly more likely to develop disease at a younger age, and dropped-head syndrome was a frequent feature. Reduced disease penetrance was evident among most affected families.

Early Expression and Cellular Localization of Proinflammatory Cytokines Interleukin-1β, Interleukin-6, and Tumor Necrosis Factor-α in Human Traumatic Spinal Cord Injury

Study Design. Post-traumatic inflammatory response was studied in 11 human cases of acute spinal cord contusion injury. Objectives. To examine the inflammatory cellular response and the immunocytochemical expression and localization of interleukin-1β, internleukin-6, and tumor necrosis factor-&agr;in human spinal cord after contusion injury. Summary of Background Data. The post-traumatic inflammatory response plays an important role in secondary injury mechanisms after spinal cord injury, and inter-leukin-1β, internleukin-6, and tumor necrosis factor-&agr; are key inflammatory mediators. Methods. The study group comprised 11 patients with spinal cord contusion injury and 2 normal individuals. Histologic and immunocytochemical assessments were undertaken to evaluate the inflammatory cellular response and the immunoexpression of interleukin-1β, internleukin-6, and tumor necrosis factor-&agr; in the injured human spinal cord. The cellular sources of interleukin-1β, internleukin-6, and tumor necrosis factor-&agr; were elucidated by immunofluorescence double-labeled confocal imaging. Results. Increased immunoreactivity of interleukin-1β, internleukin-6, and tumor necrosis factor-&agr;was detected in neurons 0.5 hour after injury, and in neurons and microglia 5 hours after injury, but the expression of these proinflammatory cytokines was short-lived and declined sharply to baseline by 2 days after injury. In the inflammatory cellular response, as early as 0.5 hour after spinal cord injury, activated microglia were detected, and axonal swellings and axons were surrounded by microglial processes. Numerous neutrophils appeared in the injured cord 1 day after injury, and then their number declined dramatically, whereas macrophages progressively increased after day 1. Conclusions Endogenous cells (neurons and microglia) in the human spinal cord, not the blood-borne leukocytes, contribute to the early production of interleukin-1β, interleukin-6, and tumor necrosis factor-&agr; in the post-traumatic inflammatory response, and microglia are involved the early response to traumatic axonal injury.

The solubility of α-synuclein in multiple system atrophy differs from that of dementia with Lewy bodies and Parkinson's disease

Intracellular inclusions containing α‐synuclein (αSN) are pathognomonic features of several neurodegenerative disorders. Inclusions occur in oligodendrocytes in multiple system atrophy (MSA) and in neurons in dementia with Lewy bodies (DLB) and Parkinsons disease (PD). In order to identify disease‐associated changes of αSN, this study compared the levels, solubility and molecular weight species of αSN in brain homogenates from MSA, DLB, PD and normal aged controls. In DLB and PD, substantial amounts of detergent‐soluble and detergent‐insoluble αSN were detected compared with controls in grey matter homogenate. Compared with controls, MSA cases had significantly higher levels of αSN in the detergent‐soluble fraction of brain samples from pons and white matter but detergent‐insoluble αSN was not detected. There was an inverse correlation between buffered saline‐soluble and detergent‐soluble levels of αSN in individual MSA cases suggesting a transition towards insolubility in disease. The differences in solubility of αSN between grey and white matter in disease may result from different processing of αSN in neurons compared with oligodendrocytes. Highly insoluble αSN is not involved in the pathogenesis of MSA. It is therefore possible that buffered saline‐soluble or detergent‐soluble forms of αSN are involved in the pathogenesis of other αSN‐related diseases.

Soluble amyloid precursor protein α reduces neuronal injury and improves functional outcome following diffuse traumatic brain injury in rats

Amyloid precursor protein (APP) has previously been shown to increase following traumatic brain injury (TBI). Whereas a number of investigators assume that increased APP may lead to the production of neurotoxic Abeta and be deleterious to outcome, the soluble alpha form of APP (sAPPalpha) is a product of the non-amyloidogenic cleavage of amyloid precursor protein that has previously been shown in vitro to have many neuroprotective and neurotrophic functions. However, no study to date has addressed whether sAPPalpha may be neuroprotective in vivo. The present study examined the effects of in vivo, posttraumatic sAPPalpha administration on functional motor outcome, cellular apoptosis, and axonal injury following severe impact-acceleration TBI in rats. Intracerebroventricular administration of sAPPalpha at 30 min posttrauma significantly improved motor outcome compared to vehicle-treated controls as assessed using the rotarod task. Immunohistochemical analysis using antibodies directed toward caspase-3 showed that posttraumatic treatment with sAPPalpha significantly reduced the number of apoptotic neuronal perikarya within the hippocampal CA3 region and within the cortex 3 days after injury compared to vehicle-treated animals. Similarly, sAPPalpha-treated animals demonstrated a reduction in axonal injury within the corpus callosum at all time points, with the reduction being significant at both 3 and 7 days postinjury. Our results demonstrate that in vivo administration of sAPPalpha improves functional outcome and reduces neuronal cell loss and axonal injury following severe diffuse TBI in rats. Promotion of APP processing toward sAPPalpha may thus be a novel therapeutic strategy in the treatment of TBI.

Severity-dependent expression of pro-inflammatory cytokines in traumatic spinal cord injury in the rat.

The post-traumatic inflammatory response in acute spinal cord contusion injury was studied in the rat. Mild and severe spinal cord injury (SCI) was produced by dropping a 10 g weight from 3 and 12 cm at the T12 vertebral level. Increased immunoreactivity of TNF-alpha in mild and severe SCI was detected in neurons at 1 h post-injury, and in neurons and microglia at 6 h post-injury, with a less significant increase in mild SCI. Expression was short-lived and declined sharply by 1 d post-injury. RT-PCR showed an early significant up-regulation of IL-1 beta, IL-6 and TNF-alpha mRNAs, maximal at 6 h post-injury with return to control levels by 24 h post-injury, the changes being less statistically significantly in mild SCI. Western blot showed early transient increases of IL-1 beta, IL-6 and TNF-alpha proteins in severe SCI but not mild SCI. Immunocytochemical, western blotting and RT-PCR analyses suggest that endogenous cells (neurons and microglia) in the spinal cord, not blood-borne leucocytes, contribute to IL-1 beta, IL-6 and TNF-alpha production in the post-traumatic inflammatory response and that their up-regulation is greater in severe than mild SCI.