Bei Ping He

Cognitive impairment in sporadic ALS A pathologic continuum underlying a multisystem disorder

Background: Traditionally considered a motor neuron–selective disorder, the clinical manifestations of ALS can include a frontotemporal dementia. Although the pathologic substrate of cognitive impairment remains to be defined, the presence of ubiquitin-immunoreactive (Ub+) intraneuronal inclusions in cortical regions has been suggested to constitute a pathologic marker of this process. Methods: The authors compared the neuropathological features of four cognitively impaired patients with ALS, four cognitively intact patients with ALS, and four neurologically normal patients. The extent and load of Ub+ neuronal inclusions, Ub+ dystrophic neurites, and superficial linear spongiosis (SLS) was determined among a number of cortical, hippocampal, and subcortical regions. Results: Although Ub+, alpha-synuclein-negative, and tau-negative neuronal inclusions were observed in both cognitively impaired and cognitively intact patients with ALS, their density and extent was greater among the former, with the difference greatest in the cingulate gyrus. Ub+ neurites were observed in a similar distribution. Only the presence of SLS, affecting the first and second cortical layers, reliably distinguished between the cognitively impaired and cognitively intact ALS subpopulations. In three of four cognitively impaired patients with ALS, SLS was associated with transcortical microglial activation, in the absence of detectable differences in astrocytosis, density of calbindin or parvalbumin neurons, or optical density of synaptophysin and SNAP-25. Conclusions: Although intraneuronal Ub+ inclusions and dystrophic neurites are observed in both ALS subpopulations, the presence of cognitive impairment was associated with a greater distribution and load of both neuropathologic features, suggesting a disease continuum. Moreover, cognitive impairment was uniformly associated with superficial linear spongiosis, a pathologic feature common to several forms of frontotemporal dementia.

Activated microglia (BV-2) facilitation of TNF-α-mediated motor neuron death in vitro

We have studied the interactions between activated microglia and injured motor neurons using an immortalized murine microglial cell line (BV-2) stimulated with either lipopolysaccharide (LPS) (Escherichia coli) or supernatant from serum-deprived motor neurons (NSC-34 cell line). Both stimuli induced BV-2 activation. Although both BV-2 supernatants induced a subsequent increase in NO generation in otherwise healthy NSC-34 cells, only LPS-activated microglial supernatant induced NSC-34 cell death through a TNF-α-dependent pathway. However, we observed a 20-fold increase in the amount of TNF-α required to kill NSC-34 cells in the absence of LPS-activated BV-2 cell supernatant, indicating that microglia secrete factor(s) that facilitate TNF-α-mediated motor neuron death in vitro.

Temporal profiles of neuronal degeneration, glial proliferation, and cell death in hNFL(+/+) and NFL(−/−)mice

Neurofilament (NF) aggregate formation within motor neurons is a pathological hallmark of both the sporadic and familial forms of amyotrophic lateral sclerosis (ALS). The relationship between aggregate formation and both microglial and astrocytic proliferation, as well as additional neuropathological features of ALS, is unknown. To examine this, we have used transgenic mice that develop NF aggregates, through either a lack of the low‐molecular‐weight NF subunit [NFL (−/−)] or the overexpression of human NFL [hNFL (+/+)]. Transgenic and wild‐type C57bl/6 mice were examined from 1 month to 18 months of age, and the temporal pattern of motor neuron degeneration, microglial and astrocytic proliferation, and heat shock protein‐70 (HSP‐70) expression characterized. We observed three overlapping phases in both transgenic mice, including transient aggregate formation, reactive microgliosis, and progressive motor neuron loss. However, only NFL (−/−) mice demonstrated significant astrogliosis and HSP‐70 upregulation in both motor neurons and astrocytes. These in vivo models suggest that the development of NF aggregates in motor neurons leads to motor neuron death, but that the interaction between the degenerating motor neurons and the adjacent non‐neuronal cells may differ significantly depending on the etiology of the NF aggregate itself.

NMDA induces NOS 1 translocation to the cell membrane in NGF-differentiated PC 12 cells.

Glutamatergic-mediated nitric oxide (NO) production occurs via the N-methyl-D-aspartic acid (NMDA) postsynaptic density protein 95 (PSD95)-neuronal nitric oxide synthase (NOS1) ternary complex. To determine whether NOS1 is targeted to the membrane subsequent to NMDA receptor activation, we examined the effect of NMDA on NOS1 subcellular localization in nerve growth factor (NGF) differentiated PC12 cells. No effect on cell viability was observed using a range of NMDA concentrations from 500 to 1000 microM. Within 3 min of stimulation with 750 microM NMDA, increased cytoplasmic NOS1 immunostaining was observed with rapid membrane staining thereafter. This was inhibited by NMDAR inhibition with MK801. This observation was confirmed using subcellular fractionation and immunoblotting. Using 4, 5-diaminofluorescein diacetate (DAF2-DA) staining and a diazotization assay, concurrent NO production was observed. When PC 12 cells were co-treated with either NMDA and N(6)-nitro-L-arginine methyl ester hydrochloride (L-NAME) or (5R, 10S)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo [a, d] cyclohepten-5, 10-imine hydrogen maleate (MK-801), nitric oxide (NO) generation was inhibited. Stimulation in a calcium-free medium did not increase NO levels. Although no evidence of cytotoxicity was observed utilizing either the MTT assay or measures of apoptosis within the maximal interval of NOS1 translocation, cell viability was reduced following 10 h of continuous NMDA exposure. While it has been shown that NMDA triggers NOS1 activation, these results indicate that NMDAR activation also mediates NOS1 targeting to the membrane. Our data validate that NGF-differentiated PC12 cells may be employed as a useful in vitro model to further study the regulation of NOS1 subsequent to NMDAR activation.

Microglia/monocytes with NG2 expression have no phagocytic function in the cortex after LPS focal injection into the rat brain

While OX42+ microglia/macrophages have been considered as a scavenger in the brain, NG2+ cells are generally considered as oligodendrocyte progenitor cells or function‐unknown glial cells. Recent evidence showed that under some pathological conditions, certain cells have become positive for both anti‐NG2 and anti‐OX42 antibodies. Our results suggested that some OX42+ microglia or macrophages were induced to express NG2 proteins 3 and 5 days later after focal injection of lipopolysaccharide into the brain cortex of Sprague‐Dawley rats. In consideration of the induction of NG2 expression may associate with gaining or losing functions of microglia/macrophages, we further showed that, while OX42+ or ED1+ microglia/macrophages presented active phagocytic function, NG2+/OX42+ cells failed to engulf latex beads. The induced expression of NG2 protein may possibly indicate the functional diversity of activated microglia/macrophages in the brain.

A morphological analysis of the motor neuron degeneration and microglial reaction in acute and chronic in vivo aluminum chloride neurotoxicity

The monthly intracisternal inoculation of aluminum chloride (AlCl3) to young adult New Zealand white rabbits induces motor neuron degeneration marked by intraneuronal neurofilamentous aggregates similar to that observed in amyotrophic lateral sclerosis (ALS). However, in contrast to ALS, this process occurs in the experimental paradigm in the absence of a glial response. In addition, whereas ALS is a fatal disorder, the cessation of aluminum exposure leads to both clinical and neuropathological recovery. Because microglia can influence neuronal regeneration, we have examined the effect of both acute and chronic aluminum exposure on microglial activation in vivo. We have studied microglial morphology in young adult New Zealand white rabbits receiving either single (1000 microg) or repeated sublethal (100 microg monthly) intracisternal inoculums of AlCl3. In addition, rabbits receiving 1000 microg AlCl3 inoculums were studied following an unilateral sciatic axotomy 48 h prior to the AlCl3 exposure. Our studies demonstrate that microglial activation in vivo is inhibited by AlCl3 exposure, and that a correlation exists between the extent of microglia suppression and the potential for recovery. This suggests that microglial activation is an important determinant of neuronal injury.

In vitro reactive nitrating species toxicity in dissociated spinal motor neurons from NFL (−/−) and hNFL (+/+) transgenic mice

We utilized fetal spinal motor neurons isolated from either NFL (−/−) or hNFL (+/+) transgenic mice to determine whether the loss of the low molecular weight neurofilament protein (NFL) places spinal motor neurons at a greater risk for cell death triggered by reactive nitrating species (RNS). After 21 days in serum‐free, antibiotic‐free medium, both the NFL (−/−) and hNFL (+/+) motor neurons developed neurofilamentous aggregates. Cultures were then exposed to nitric oxide (100 μM NOC 5, 100 μM NOC 12, or 2 mM sodium nitroprusside) or to peroxynitrite (250 mM SIN‐1) for varying intervals. NFL (−/−) cultures demonstrated extensive numbers of apoptotic neurons within six hours and complete cell loss by 24 hours in response to NOC 5 and NOC 12. In contrast, apoptosis was only observed in the motor neurons derived from control (C57bl/6) or hNLF (+/+) mice at 24 hours. In response to 2 mM sodium nitroprusside, necrosis was induced in all cells within 60 minutes. In response to 250 mM SIN‐1, both C57bl/6 and hNFL (+/+) cells survived to six hours with only minimal evidence of degeneration while NFL (−/−) motor neurons were necrotic by 60 minutes. These observations suggest that NFL deficient motor neurons are at an enhanced risk of cell death mediated by RNS.

The activation of NG2 expressing cells is downstream to microglial reaction and mediated by the transforming growth factor beta 1

In the present study, we investigated the mechanism of activation of NG2 expressing cells. Application of microglial inhibitors not only attenuated morphological changes but also significantly retarded increase in the number of NG2 expressing cells. Intracerebral injection of TGF-β1 led to a profound activation of NG2 glia as well as an earlier accumulation of NG2(+)-microglia, whilst inhibition of TGF-β1 Smad2/3 signalling pathway eventually attenuated their active responses. We conclude that the activation of NG2 expressing cells is an event downstream to microglial reaction and TGF-β1 secreted from microglia might play an important role in modulation of the function of NG2 expressing cells.