Kirat K. Chand

Loss of β2‐laminin alters calcium sensitivity and voltage‐gated calcium channel maturation of neurotransmission at the neuromuscular junction

Neuromuscular junctions from β2‐laminin‐deficient mice exhibit lower levels of calcium sensitivity. Loss of β2‐laminin leads to a failure in switching from N‐ to P/Q‐type voltage‐gated calcium channel (VGCC)‐mediated transmitter release that normally occurs with neuromuscular junction maturation. The motor nerve terminals from β2‐laminin‐deficient mice fail to up‐regulate the expression of P/Q‐type VGCCs clusters and down‐regulate N‐type VGCCs clusters, as they mature. There is decreased co‐localisation of presynaptic specialisations in β2‐laminin‐deficient neuromuscular junctions as a consequence of lesser P/Q‐type VGCC expression. These findings support the idea that β2‐laminin is critical in the organisation and maintenance of active zones at the neuromuscular junction via its interaction with P/Q‐type VGCCs, which aid in stabilisation of the synapse.

Review: Neuroinflammation in intrauterine growth restriction

Disruption to the maternal environment during pregnancy from events such as hypoxia, stress, toxins, inflammation, and reduced placental blood flow can affect fetal development. Intrauterine growth restriction (IUGR) is commonly caused by chronic placental insufficiency, interrupting supply of oxygen and nutrients to the fetus resulting in abnormal fetal growth. IUGR is a major cause of perinatal morbidity and mortality, occurring in approximately 5-10% of pregnancies. The fetal brain is particularly vulnerable in IUGR and there is an increased risk of long-term neurological disorders including cerebral palsy, epilepsy, learning difficulties, behavioural difficulties and psychiatric diagnoses. Few studies have focused on how growth restriction interferes with normal brain development in the IUGR neonate but recent studies in growth restricted animal models demonstrate increased neuroinflammation. This review describes the role of neuroinflammation in the progression of brain injury in growth restricted neonates. Identifying the mediators responsible for alterations in brain development in the IUGR infant is key to prevention and treatment of brain injury in these infants.

Tick holocyclotoxins trigger host paralysis by presynaptic inhibition

Ticks are important vectors of pathogens and secreted neurotoxins with approximately 69 out of 692 tick species having the ability to induce severe toxicoses in their hosts. The Australian paralysis tick (Ixodes holocyclus) is known to be one of the most virulent tick species producing a flaccid paralysis and fatalities caused by a family of neurotoxins known as holocyclotoxins (HTs). The paralysis mechanism of these toxins is temperature dependent and is thought to involve inhibition of acetylcholine levels at the neuromuscular junction. However, the target and mechanism of this inhibition remain uncharacterised. Here, we report that three members of the holocyclotoxin family; HT-1 (GenBank AY766147), HT-3 (GenBank KP096303) and HT-12 (GenBank KP963967) induce muscle paralysis by inhibiting the dependence of transmitter release on extracellular calcium. Previous study was conducted using extracts from tick salivary glands, while the present study is the first to use pure toxins from I. holocyclus. Our findings provide greater insight into the mechanisms by which these toxins act to induce paralysis.

Functional decline at the aging neuromuscular junction is associated with altered laminin-α4 expression

Laminin-α4 is involved in the alignment of active zones to postjunctional folds at the neuromuscular junction (NMJ). Prior study has implicated laminin-α4 in NMJ maintenance, with altered NMJ morphology observed in adult laminin-α4 deficient mice (lama4−/−). The present study further investigated the role of laminin-α4 in NMJ maintenance by functional characterization of transmission properties, morphological investigation of synaptic proteins including synaptic laminin-α4, and neuromotor behavioral testing. Results showed maintained perturbed transmission properties at lama4−/− NMJs from adult (3 months) through to aged (18-22 months). Hind-limb grip force demonstrated similar trends as transmission properties, with maintained weaker grip force across age groups in lama4−/−. Interestingly, both transmission properties and hind-limb grip force in aged wild-types resembled those observed in adult lama4−/−. Most significantly, altered expression of laminin-α4 was noted at the wild-type NMJs prior to the observed decline in transmission properties, suggesting that altered laminin-α4 expression precedes the decline of neurotransmission in aging wild-types. These findings significantly support the role of laminin-α4 in maintenance of the NMJ during aging.

Neonatal seizures are associated with redistribution and loss of GABAA α‐subunits in the hypoxic‐ischaemic pig

Seizures are a common manifestation of hypoxic‐ischaemic brain injury in the neonate. In status epilepticus models alterations to GABAAR subunit expression have been suggested to contribute to (i) abnormal development of the GABAergic system, (ii) why seizures become self‐sustaining and (iii) the development of pharmacoresistance. Detailed investigation of GABAAR subunit protein expression after neonatal hypoxia‐ischaemia (HI) is currently insufficient. Using our pig model of HI and subsequent spontaneous neonatal seizures, we investigated changes in protein expression of the three predominant α‐subunits of the GABAAR; α1, α2 and α3. Anaesthetized, ventilated newborn pigs (< 24 h old) were subjected to 30 min HI and subsequently recovered to 24 or 72 h. Amplitude‐integrated electroencephalography was used to monitor brain activity and identify seizure activity. Brain tissue was collected post‐mortem and GABAAR α‐subunit protein expression was analysed using western blot and immunohistochemistry. GABAAR α1 and α3 protein expression was significantly reduced in animals that developed seizures after HI; HI animals that did not develop seizures did not exhibit the same reductions. Immunohistochemistry revealed decreased α1 and α3 expression, and α1 redistribution from the cell membrane to the cytosol, in the hippocampus of seizure animals. Multivariate analyses, controlling for HI severity and neuronal injury, revealed that seizures were independently associated with significant GABAAR α3 reduction. This is the first study to show loss and redistribution of GABAAR α‐subunits in a neonatal brain experiencing seizures. Our findings are similar to those reported in models of SE and in chronic epilepsy.

Defects in synaptic transmission at the neuromuscular junction precede motor deficits in a TDP-43Q331K transgenic mouse model of amyotrophic lateral sclerosis

Transactive response DNA‐binding protein‐43 (TDP‐43) is involved in gene regulation via the control of RNA transcription, splicing, and transport. TDP‐43 is a major protein component of ubiquinated inclusions that are found in amyotrophic lateral sclerosis (ALS); however, the function of TDP‐43 at the neuromuscular junction (NMJ) and its role in ALS pathogenesis is largely unknown. Here, we show that TDP‐43Q331K mutation in mice resulted in impaired neurotransmission by age 3 mo, preceding deficits in motor function and motor neuron loss, which were observed from age 10 mo. These defects were in the effective fusion and release of synaptic vesicles within the motor nerve terminal and manifested in decreased quantal content and reduced probability of quantal release. We observed morphologic alterations that were associated with the TDP‐43Q331K mutation, such as aberrant innervation patterns and the distribution of synaptic vesicle‐related proteins, which is indicative of a failing NMJ undergoing synaptic remodeling. These findings support a growing acceptance that dysregulation of the NMJ function is a key early event in the pathology of ALS.—Chand, K. K., Lee, K. M., Lee, J. D, Qiu, H., Willis, E. F., Lavidis, N. A., Hilliard, M. A., Noakes, P. G. Defects in synaptic transmission at the neuromuscular junction precede motor deficits in a TDP‐43Q331K transgenic mouse model of amyotrophic lateral sclerosis. FASEB J. 32, 2676–2689 (2018). www.fasebj.org

Loss of laminin-α4 results in pre- and postsynaptic modifications at the neuromuscular junction

Synaptic basal lamina such as laminin‐421 (α4β2γ1) mediate differentiation of the neuromuscular junction (NMJ). Laminins interact with their pre‐ or postsynaptic receptors to provide stability and alignment of the pre‐ to postsynaptic specializations. Knockout of the laminin‐α4 gene (lama4) does not alter gross NMJ morpho‐genesis. However, mice deficient in laminin‐α4 (lama4−/− mice) display disruptions in the alignment of the active zones and postsynaptic folds at the NMJ, although the physiological consequences of this loss have not been examined. The present study investigated the differences in neurotransmission during the early development and maturation of the NMJ in lama4−/− and wild‐type mice. Lama4−/− NMJs demonstrated a decrease in miniature end‐plate potential (EPP) frequency and increased amplitude of miniature EPPs and evoked EPPs. Binomial parameters analysis of neurotransmitter release revealed a decrease in quantal release, the result of a decrease in the number of active release sites, but not in the probability of transmitter release. Lama4−/− NMJs displayed higher levels of synaptic depression under high‐frequency stimulation and altered facilitation, suggesting compromised delivery of synaptic vesicles. This idea is supported by our molecular investigations of lama4−/− NMJs, where we see altered distribution of Bassoon, a molecular component of active zones, presumably resulting from perturbed neurotransmission. —Chand, K. K., Lee, K. M., Lavidis, N. A., Noakes, P. G. Loss of laminin‐α4 results in pre‐ and postsynaptic modifications at the neuromuscular junction. FASEB J. 31, 1323–1336 (2017) www.fasebj.org

Therapeutic potential to reduce brain injury in growth restricted newborns

Brain injury in intrauterine growth restricted (IUGR) infants is a major contributing factor to morbidity and mortality worldwide. Adverse outcomes range from mild learning difficulties, to attention difficulties, neurobehavioral issues, cerebral palsy, epilepsy, and other cognitive and psychiatric disorders. While the use of medication to ameliorate neurological deficits in IUGR neonates has been identified as warranting urgent research for several years, few trials have been reported. This review summarises clinical trials focusing on brain protection in the IUGR newborn as well as therapeutic interventions trialled in animal models of IUGR. Therapeutically targeting mechanisms of brain injury in the IUGR neonate is fundamental to improving long‐term neurodevelopmental outcomes. Inflammation is a key mechanism in neonatal brain injury; and therefore an appealing target. Ibuprofen, an anti‐inflammatory drug currently used in the preterm neonate, may be a potential therapeutic candidate to treat brain injury in the IUGR neonate. To better understand the potential of ibuprofen and other therapeutic agents to be neuroprotective in the IUGR neonate, long‐term follow‐up information of neurodevelopmental outcomes must be studied. Where agents such as ibuprofen are shown to be effective, have a good safety profile and are relatively inexpensive, they can be widely adopted and lead to improved outcomes.

Disruption to the 5-HT7 Receptor Following Hypoxia–Ischemia in the Immature Rodent Brain

It has become increasingly evident the serotonergic (5-hydroxytryptamine, 5-HT) system is an important central neuronal network disrupted following neonatal hypoxic–ischemic (HI) insults. Serotonin acts via a variety of receptor subtypes that are differentially associated with behavioural and cognitive mechanisms. The 5-HT7 receptor is purported to play a key role in epilepsy, anxiety, learning and memory and neuropsychiatric disorders. Furthermore, the 5-HT7 receptor is highly localized in brain regions damaged following neonatal HI insults. Utilising our well-established neonatal HI model in the postnatal day 3 (P3) rat pup we demonstrated a significant decrease in levels of the 5-HT7 protein in the frontal cortex, thalamus and brainstem one week after insult. We also observed a relative decrease in both the cytosolic and membrane fractions of 5-HT7. The 5-HT7 receptor was detected on neurons throughout the cortex and thalamus, and 5-HT cell bodies in the brainstem. However we found no evidence of 5-HT7 co-localisation on microglia or astrocytes. Moreover, minocycline treatment did not significantly prevent the HI-induced reductions in 5-HT7. In conclusion, neonatal HI injury caused significant disruption to 5-HT7 receptors in the forebrain and brainstem. Yet the use of minocycline to inhibit activated microglia, did not prevent the HI-induced changes in 5-HT7 expression.