Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

University of South Carolina

Long-term primary neuronal cultures are a useful tool for the investigation of biochemical processes associated with neuronal senescence. Improvements in available technology make it possible to observe maturation of neural cells isolated from different regions of the rodent brain over a prolonged period in vitro. Existing experimental evidence suggests that cellular aging occurs in mature, long-term, primary neuronal cell cultures. However, detailed studies of neuronal development in vitro are needed to demonstrate the validity of long-term cell culture-based models for investigation of the biochemical mechanisms of in vitro neuronal development and senescence.

Endogenous amyloidogenesis in long-term rat hippocampal cell cultures.

HIV-1 enters the central nervous system early in infection; although HIV-1 does not directly infect neurons, HIV-1 may cause a variety of neurological disorders. Neuronal loss has been found in HIV-1, but synaptodendritic injury is more closely associated with the neurocognitive disorders of HIV-1. The HIV-1 transactivator of transcription (Tat) protein causes direct and indirect damage to neurons. The cysteine rich domain (residues 22-37) of Tat is important for producing neuronal death; however, little is known about the effects of the Tat protein functional domains on the dendritic network. The ability of HIV-1 Tat 1-101 Clades B and C, Tat 1-86 and Tat 1-72 proteins, as well as novel peptides (truncated 47-57, 1-72δ31-61, and 1-86 with a mutation at Cys22) to produce early synaptodendritic injury (24h), relative to later cell death (48h), was examined using cell culture. Treatment of primary hippocampal neurons with Tat proteins 1-72, 1-86 and 1-101B produced a significant early reduction in F-actin labeled puncta, implicating that these peptides play a role in synaptodendritic injury. Variants with a mutation, deletion, or lack of a cysteine rich region (1-86[Cys22], 1-101C, 1-72δ31-61, or 47-57) did not cause a significant reduction in F-actin rich puncta. Tat 1-72, 1-86, and 1-101B proteins did not significantly differ from one another, indicating that the second exon (73-86 or 73-101) does not play a significant role in the reduction of F-actin puncta. Conversely, peptides with a mutation, deletion, or lack of the cysteine rich domain (22-37) failed to produce a loss of F-actin puncta, indicating that the cysteine rich domain plays a key role in synaptodendritic injury. Collectively, these results suggest that for Tat proteins, 1) synaptodendritic injury occurs early, relative to cell death, and 2) the cysteine rich domain of the first exon is key for synaptic loss. Preventing such early synaptic loss may attenuate HIV-1 associated neurocognitive disorders.

HIV-1 Tat protein variants: critical role for the cysteine region in synaptodendritic injury.

HIV-1 infects the brain and, despite antiretroviral therapy, many infected individuals suffer from HIV-1-associated neurocognitive disorders (HAND). HAND is associated with dendritic simplification and synaptic loss. Prevention of synaptodendritic damage may ameliorate or forestall neurocognitive decline in latent HIV-1 infections. The HIV-1 transactivating protein (Tat) is produced during viral latency in the brain and may cause synaptodendritic damage. This study examined the integrity of the dendritic network after exposure to HIV-1 Tat by labeling filamentous actin (F-actin)-rich structures (puncta) in primary neuronal cultures. After 24 h of treatment, HIV-1 Tat was associated with the dendritic arbor and produced a significant reduction of F-actin-labeled dendritic puncta as well as loss of dendrites. Pre-treatment with either of two plant-derived phytoestrogen compounds (daidzein and liquiritigenin), significantly reduced synaptodendritic damage following HIV-1 Tat treatment. In addition, 6 days after HIV-1 Tat treatment, treatment with either daidzein, or liquiritigenin enhanced recovery, via the estrogen receptor, from HIV-1 Tat-induced synaptodendritic damage. These results suggest that either liquiritigenin or daidzein may not only attenuate acute synaptodendritic injury in HIV-1 but may also promote recovery from synaptodendritic damage. The HIV-1 transactivating protein (Tat) is produced during viral latency in the brain. Treatment with either daidzein or liquiritigenin restored the loss of synaptic connectivity produced by HIV-1 Tat. This neurorestoration was mediated by estrogen receptors (ER). These results suggest that plant-derived phytoestrogens may promote recovery from HIV-1-induced synaptodendritic damage.

Synaptodendritic recovery following HIV Tat exposure: neurorestoration by phytoestrogens.

Illicit drugs, such as cocaine, are known to increase the likelihood and severity of HIV-1 associated neurocognitive disorders (HAND). In the current studies synaptic integrity was assessed following exposure to low concentrations of the HIV-1 viral protein Tat 1-86B, with or without cocaine, by quantifying filamentous actin (F-actin) rich structures (i.e., puncta and dendritic spines) on neuronal dendrites in vitro. In addition, the synapse-protective effects of either R-Equol (RE) or S-Equol (SE; derivatives of the soy isoflavone, daidzein) were determined. Individually, neither low concentrations of HIV-1 Tat (10 nM) nor low concentrations of cocaine (1.6 μM) had any significant effect on F-actin puncta number; however, the same low concentrations of HIV-1 Tat + cocaine in combination significantly reduced dendritic synapses. This synaptic reduction was prevented by pre-treatment with either RE or SE, in an estrogen receptor beta dependent manner. In sum, targeted therapeutic intervention with SE may prevent HIV-1 + drug abuse synaptopathy, and thereby potentially influence the development of HAND.

Sarah J. Bertrand

Laboratory Program in Behavioral,
Neuroscience,
Department of Psychology,
Laboratory Program in Behavioral, Neuroscience, Department of Psychology

University of South Carolina

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Sarah J. Bertrand

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory Program in Behavioral,Neuroscience,Department of Psychology,Laboratory Program in Behavioral, Neuroscience, Department of Psychology

University of South Carolina

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Laboratory Program in Behavioral,
Neuroscience,
Department of Psychology,
Laboratory Program in Behavioral, Neuroscience, Department of Psychology