Haemoglobin neurotoxicity, haptoglobin scavenging and synaptic function in subarachnoid haemorrhage

During subarachnoid haemorrhage, blood spreads into the subarachnoid space and surrounding tissues. Slow release of haemoglobin (Hb) from red blood cell lysis causes oxidative damage and cell death. High rates of disability and cognitive decline in SAH survivors is attributed to loss of neurons and functional connections during secondary brain injury. Haptoglobin (Hp) sequesters free Hb for clearance, but this scavenging system is overwhelmed in a haemorrhage. Hp infusion has been shown to attenuate cytotoxic effects of haemoglobin on neurons in both in vitro and in vivo models of SAH, and other haemorrhagic conditions. The functional effects of clinically relevant and sub-lethal Hb concentrations on surviving neurons, and whether cellular function can be protected with Hp treatment, remain unclear.In this thesis, the effects of a one week exposure to Hb are analysed in primary hippocampal neuron cultures. Our results demonstrate reduced ATP levels and neurite beading at a range of concentrations of haemolysate. At a sub-lethal concentration of 10 μM free Hb, which is the average Hb peak in the CSF after SAH in humans, intrinsic membrane properties are normal. However, we found a reduction in AMPA receptor-mediated current amplitude in the absence of presynaptic alterations, indicating fewer AMPA receptors at the synapse, and an overall reduction in GluA1 subunit expression. By scavenging one-third of free Hb with Hp in vitro, synaptic AMPA receptor impairment can be partially rescued and is indistinguishable from control, indicating that functional rescue can be achieved by Hp even without 1:1 stoichiometric neutralization of Hb. At higher concentrations of free Hb, higher levels of saturation with Hp can protect from ATP deficits and neurite beading. Hp in itself does not alter pre- or post-synaptic measures of synaptic neurotransmission, and has no effect on intrinsic membrane properties at 10μM. Our data highlight a role for Hb in modifying synaptic function after SAH, which may link to impaired cognition or plasticity, and the potential of haptoglobin as a therapy for subarachnoid haemorrhage

Dataset in support of the Southampton doctoral thesis 'Haemoglobin neurotoxicity, haptoglobin scavenging and synaptic function in subarachnoid haemorrhage'

File of all experimental data analysed for the thesis: Haemoglobin Neurotoxicity, Haptoglobin Scavenging and Synaptic Function in Subarachnoid Haemorrhage.</span

A luminescence-based reporter to study tau secretion reveals overlapping mechanisms for the release of healthy and pathological tau

In Alzheimer’s disease, tau pathology is thought to spread via a prion-like manner along connected neuronal networks. For this to occur, the usually cytosolic tau protein must be secreted via an unconventional mechanism prior to uptake into the connected neuron. While secretion of healthy and pathological tau has been documented, it remains under-investigated whether this occurs via overlapping or distinct processes. Here, we established a sensitive bioluminescence-based assay to assess mechanisms underlying the secretion of pseudohyperphosphorylated and wild-type tau in cultured murine hippocampal neurons. We found that under basal conditions, both wild-type and mutant tau are secreted, with mutant tau being more robustly secreted. Pharmacological stimulation of neuronal activity led to a modest increase of wild-type and mutant tau secretion, whereas inhibition of activity had no effect. Interestingly, inhibition of heparin sulfate proteoglycan (HSPG) biosynthesis drastically decreased secretion of both wild-type and mutant tau without affecting cell viability. This shows that native and pathological tau share release mechanisms; both activity-dependent and non-activity-dependent secretion of tau is facilitated by HSPGs

A primate-specific short GluN2A-NMDA receptor isoform is expressed in the human brain

Glutamate receptors of the N-methyl-D-aspartate (NMDA) family are coincident detectors of pre- and postsynaptic activity, allowing Ca2+ influx into neurons. These properties are central to neurological disease mechanisms and are proposed to be the basis of associative learning and memory. In addition to the well-characterised canonical GluN2A NMDAR isoform, large-scale open reading frames in human tissues had suggested the expression of a primate-specific short GluN2A isoform referred to as GluN2A-S. Here, we confirm the expression of both GluN2A transcripts in human and primate but not rodent brain tissue, and show that they are translated to two corresponding GluN2A proteins present in human brain. Furthermore, we demonstrate that recombinant GluN2A-S co-assembles with the obligatory NMDAR subunit GluN1 to form functional NMDA receptors. These findings suggest a more complex NMDAR repertoire in human brain than previously thought.<br/