Dawn H. W. Lau

A strategy for constructing multi-payload thio-antibody conjugates using pyridazinedione scaffolds, para -anisidine derivatives, and various click chemistries.

Neuroinflammation and hyperexcitability have been implicated in the pathogenesis of neurodegenerative disease, and new models are required to investigate the cellular crosstalk involved in these processes. We developed an approach to generate a quantitative and reproducible triculture system that is suitable for pharmacological studies. While primary rodent cells were previously grown in a coculture medium formulated to support only neurons and astrocytes, we now optimised a protocol to generate tricultures containing neurons, astrocytes and microglia by culturing in a medium designed to support all three cell types and adding exogenous microglia to cocultures. Immunocytochemistry was used to confirm the intended cell types were present. The percentage of ramified microglia in the tricultures decreases as the number of microglia present increases. Multi-electrode array (MEA) recordings indicate that microglia in the triculture model suppress neuronal activity in a dose-dependent manner. Neurons in both cocultures and tricultures were responsive to the potassium channel blocker 4-aminopyridine (4-AP) suggesting that neurons remained viable and functional in the triculture model. Furthermore, suppressed neuronal activity in tricultures correlated with decreased densities of dendritic spines and of the postsynaptic protein Homer1 along dendrites, indicative of a direct or indirect effect of microglia on synapse function. We thus present a functional triculture model, which, due to its more complete cellular composition, is a more relevant model than standard cocultures. The model can be used to probe glia-neuron interactions and subsequently aid the development of assays for drug discovery, using neuronal excitability as a functional endpoint.

Damage to axonal transport is an early pathogenic event in Alzheimer’s disease. The amyloid precursor protein (APP) is a key axonal transport cargo since disruption to APP transport promotes amyloidogenic processing of APP. Moreover, altered APP processing itself disrupts axonal transport. The mechanisms that regulate axonal transport of APP are therefore directly relevant to Alzheimer’s disease pathogenesis. APP is transported anterogradely through axons on kinesin-1 motors and one route for this transport involves calsyntenin-1, a type-1 membrane spanning protein that acts as a direct ligand for kinesin-1 light chains (KLCs). Thus, loss of calsyntenin-1 disrupts APP axonal transport and promotes amyloidogenic processing of APP. Phosphorylation of KLC1 on serine-460 has been shown to reduce anterograde axonal transport of calsyntenin-1 by inhibiting the KLC1-calsyntenin-1 interaction. Here we demonstrate that in Alzheimer’s disease frontal cortex, KLC1 levels are reduced and the relative levels of KLC1 serine-460 phosphorylation are increased; these changes occur relatively early in the disease process. We also show that a KLC1 serine-460 phosphomimetic mutant inhibits axonal transport of APP in both mammalian neurons in culture and inDrosophilaneurons in vivo. Finally, we demonstrate that expression of the KLC1 serine-460 phosphomimetic mutant promotes amyloidogenic processing of APP. Together, these results suggest that increased KLC1 serine-460 phosphorylation contributes to Alzheimer’s disease.

Cyclin dependent kinase-5 (cdk5)/p35 is a neuronal kinase that regulates key axonal and synaptic functions but the mechanisms by which it is transported to these locations are unknown. Lemur tyrosine kinase-2 (LMTK2) is a binding partner for p35 and here we show that LMTK2 also interacts with kinesin-1 light chains (KLC1/2). Binding to KLC1/2 involves a C-terminal tryptophan/aspartate (WD) motif in LMTK2 and the tetratricopeptide repeat (TPR) domains in KLC1/2, and this interaction facilitates axonal transport of LMTK2. Thus, siRNA loss of KLC1 or mutation of the WD motif disrupts axonal transport of LMTK2. We also show that LMTK2 facilitates the formation of a complex containing KLC1 and p35 and that siRNA loss of LMTK2 disrupts axonal transport of both p35 and cdk5. Finally, we show that LMTK2 levels are reduced in Alzheimer’s disease brains. Damage to axonal transport and altered cdk5/p35 are pathogenic features of Alzheimer’s disease. Thus, LMTK2 binds to KLC1 to direct axonal transport of p35 and its loss may contribute to Alzheimer’s disease.

Signaling between the endoplasmic reticulum (ER) and mitochondria regulates a number of key neuronal functions. This signaling involves close physical contacts between the two organelles that are mediated by “tethering proteins” that function to recruit regions of ER to the mitochondrial surface. The ER protein, vesicle-associated membrane protein-associated protein B (VAPB) and the mitochondrial membrane protein, protein tyrosine phosphatase interacting protein-51 (PTPIP51), interact to form one such tether. Recently, damage to ER-mitochondria signaling involving disruption of the VAPB-PTPIP51 tethers has been linked to the pathogenic process in Parkinson’s disease, fronto-temporal dementia (FTD) and related amyotrophic lateral sclerosis (ALS). Loss of neuronal synaptic function is a key feature of Parkinson’s disease and FTD/ALS but the roles that ER-mitochondria signaling and the VAPB-PTPIP51 tethers play in synaptic function are not known. Here, we demonstrate that the VAPB-PTPIP51 tethers regulate synaptic activity. VAPB and PTPIP51 localise and form contacts at synapses, and stimulating neuronal activity increases ER-mitochondria contacts and the VAPB-PTPIP51 interaction. Moreover, siRNA loss of VAPB or PTPIP51 perturbs synaptic function and dendritic spine morphology. Our results reveal a new role for the VAPB-PTPIP51 tethers in neurons and suggest that damage to ER-mitochondria signaling contributes to synaptic dysfunction in Parkinson’s disease and FTD/ALS.

Fronto-temporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are two related and incurable neurodegenerative diseases. Features of these diseases include pathological protein inclusions in affected neurons with TAR DNA-binding protein 43 (TDP-43), dipeptide repeat proteins derived from the C9ORF72 gene, and fused in sarcoma (FUS) representing major constituent proteins in these inclusions. Mutations in C9ORF72 and the genes encoding TDP-43 and FUS cause familial forms of FTD/ALS which provides evidence to link the pathology and genetics of these diseases. A large number of seemingly disparate physiological functions are damaged in FTD/ALS. However, many of these damaged functions are regulated by signalling between the endoplasmic reticulum and mitochondria, and this has stimulated investigations into the role of endoplasmic reticulum-mitochondria signalling in FTD/ALS disease processes. Here, we review progress on this topic.

Defective FUS metabolism is strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), but the mechanisms linking FUS to disease are not properly understood. However, many of the functions disrupted in ALS/FTD are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling is facilitated by close physical associations between the two organelles that are mediated by binding of the integral ER protein VAPB to the outer mitochondrial membrane protein PTPIP51, which act as molecular scaffolds to tether the two organelles. Here, we show that FUS disrupts the VAPB–PTPIP51 interaction and ER–mitochondria associations. These disruptions are accompanied by perturbation of Ca ²⁺ uptake by mitochondria following its release from ER stores, which is a physiological read‐out of ER–mitochondria contacts. We also demonstrate that mitochondrial ATP production is impaired in FUS‐expressing cells; mitochondrial ATP production is linked to Ca ²⁺ levels. Finally, we demonstrate that the FUS‐induced reductions to ER–mitochondria associations and are linked to activation of glycogen synthase kinase‐3β (GSK‐3β), a kinase already strongly associated with ALS/FTD.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterised by neuropathological deposits of amyloid plaques and neurofibrillary tangles comprised of β-amyloid and tau protein, respectively. In AD, tau becomes abnormally phosphorylated and aggregates to form intracellular deposits. However, the mechanisms by which tau exerts neurotoxicity in disease remain unclear. Recent studies have suggested that the presence of tau at synapses may indicate a role in neuronal signalling, which could be disrupted in pathological conditions. The non-receptor-associated tyrosine kinase fyn is located at the dendrite in neurons, where it was recently shown to interact with tau to stabilise receptor complexes at the post-synaptic density. Fyn also co-localises with tau in a proportion of neurons containing tau tangles in AD and fyn is also a tau kinase. Hence, tau-fyn interactions could play a pathogenic role in AD. Here we report the identification of critical proline residues, Pro213, Pro216, and Pro219, located within the fifth and sixth Pro-X-X-Pro motifs in the proline-rich region of tau, that are important for its binding to fyn. These residues in tau are flanked by numerous phosphorylation sites and therefore we investigated the relationship between fyn and the degree of tau phosphorylation in human post-mortem brain tissue. We found no difference in the amount of fyn present in control and AD brain. Notably, however, there was a significant correlation between fyn and phosphorylated tau at specific phospho-epitopes in control, but not in AD brain. Our results suggest that the pathological mechanisms underlying AD, that result in increased tau phosphorylation, may disrupt the physiological relationship between tau phosphorylation and fyn.