TDP-43 pathology links innate and adaptive immunity in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is the most common fatal motor neuron disease. Approximately 90% of ALS patients exhibit pathology of the master RNA regulator, Transactive Response DNA Binding protein (TDP-43). Despite the prevalence TDP-43 pathology in ALS motor neurons, recent findings suggest immune dysfunction is a determinant of disease progression in patients. Whether TDP-43 pathology elicits disease-modifying immune responses in ALS remains underexplored. In this study, we demonstrate that TDP-43 pathology is internalized by antigen presenting cells, causes vesicle rupture, and leads to innate and adaptive immune cell activation. Using a multiplex imaging platform, we observed interactions between innate and adaptive immune cells near TDP-43 pathological lesions in ALS brain. We used a mass cytometry-based whole-blood stimulation assay to provide evidence that ALS patient peripheral immune cells exhibit responses to TDP-43 aggregates. Taken together, this study provides a novel link between TDP-43 pathology and ALS immune dysfunction, and further highlights the translational and diagnostic implications of monitoring and manipulating the ALS immune response.

Activity-dependent tau cleavage by caspase-3 promotes neuronal dysfunction and synaptotoxicity.

Tau-mediated toxicity is associated with cognitive decline and Alzheimer's disease (AD) progression. In particular, tau post-translational modifications (PTMs) are thought to generate aberrant tau species resulting in neuronal dysfunction. Despite being well characterized in postmortem AD brain, it is unclear how caspase-mediated C-terminal tau cleavage promotes neurodegeneration, as few studies have developed the models to dissect this pathogenic mechanism. Here, we show that proteasome impairment results in cleaved tau accumulation at the post-synaptic density (PSD), a process that is modulated by neuronal activity. Cleaved tau (at residue D421) impairs neuronal firing and causes inefficient initiation of network bursts, consistent with reduced excitatory drive. We propose that reduced neuronal activity, or silencing, is coupled to proteasome dysfunction, which drives cleaved tau accumulation at the PSD and subsequent synaptotoxicity. Our study connects three common themes in the progression of AD: impaired proteostasis, caspase-mediated tau cleavage, and synapse degeneration.

N-Phenyl-1-(phenylsulfonyl)-1H-1,2,4-triazol-3-amine as a New Class of HIV-1 Non-nucleoside Reverse Transcriptase Inhibitor.

Highly active antiretroviral therapy (HAART) has revolutionized human immunodeficiency virus (HIV) healthcare, turning it from a terminal to a potentially chronic disease, although some patients can develop severe comorbidities. These include neurological complications, such as HIV-associated neurocognitive disorders (HAND), which result in cognitive and/or motor function symptoms. We now describe the discovery, synthesis, and evaluation of a new class of N-phenyl-1-(phenylsulfonyl)-1H-1,2,4-triazol-3-amine HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTI) aimed at avoiding HAND. The most promising molecule, 12126065, exhibited antiviral activity against wild-type HIV-1 in TZM cells (EC50 = 0.24 nM) with low in vitro cytotoxicity (CC50 = 4.8 µM) as well as retained activity against clinically relevant HIV mutants. 12126065 also demonstrated no in vivo acute or subacute toxicity, good in vivo brain penetration, and minimal neurotoxicity in mouse neurons up to 10 µM, with a 50% toxicity concentration (TC50) of >100 µM, well below its EC50.

Tandem detergent-extraction and immunoprecipitation of proteinopathy: Scalable enrichment of ALS-associated TDP-43 aggregates.

Transactive response DNA-binding protein of 43 kDa (TDP-43) is a highly conserved, ubiquitously expressed nucleic acid-binding protein that regulates DNA/RNA metabolism. Genetics and neuropathology studies have linked TDP-43 to several neuromuscular and neurological disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Under pathological conditions, TDP-43 mislocalizes to the cytoplasm where it forms insoluble, hyper-phosphorylated aggregates during disease progression. Here, we optimized a scalable in vitro immuno-purification strategy referred to as tandem detergent-extraction and immunoprecipitation of proteinopathy (TDiP) to isolate TDP-43 aggregates that recapitulate those identified in postmortem ALS tissue. Moreover, we demonstrate that these purified aggregates can be utilized in biochemical, proteomics, and live-cell assays. This platform offers a rapid, accessible, and streamlined approach to study ALS disease mechanisms, while overcoming many limitations that have hampered TDP-43 disease modeling and therapeutic drug discovery efforts.

Association of Serum Pyridoxal Phosphate Levels with Established Status Epilepticus.

BACKGROUND: The objective of this study was to determine the prevalence of pyridoxine deficiency, measured by pyridoxal phosphate (PLP) levels, in patients admitted to the hospital with established (benzodiazepine-resistant) status epilepticus (SE) (eSE) and to compare to three control groups: intensive care unit (ICU) patients without SE (ICU-noSE), non-ICU inpatients without SE (non-ICU), and outpatients with or without a history of epilepsy (outpatient). METHODS: This retrospective cohort study was conducted at the University of North Carolina Hospitals and Yale New Haven Hospital. Participants included inpatients and outpatients who had serum PLP levels measured during clinical care between January 2018 and March 2021. The first PLP level obtained was categorized as normal (> 30 nmol/L), marginal (≤ 30 nmol/L), deficient (≤ 20 nmol/L), and severely deficient (≤ 5 nmol/L). RESULTS: A total of 293 patients were included (52 eSE, 40 ICU-noSE, 44 non-ICU, and 157 outpatient). The median age was 55 (range 19-99) years. The median PLP level of the eSE group (12 nmol/L) was lower than that of the ICU-noSE (22 nmol/L, p = 0.003), non-ICU (16 nmol/L, p = 0.05), and outpatient groups (36 nmol/L, p < 0.001). Patients with eSE had a significantly higher prevalence of marginal and deficient PLP levels (90 and 80%, respectively) than patients in each of the other three groups (ICU-noSE: 70, 50%; non-ICU: 63, 54%; outpatient: 38, 21%). This significantly higher prevalence persisted after correcting for critical illness severity and timing of PLP level collection. CONCLUSIONS: Our study confirms previous findings indicating a high prevalence of pyridoxine deficiency (as measured by serum PLP levels) in patients with eSE, including when using a more restricted definition of pyridoxine deficiency. Prevalence is higher in patients with eSE than in patients in all three control groups (ICU-noSE, non-ICU, and outpatient). Considering the role of pyridoxine, thus PLP, in the synthesis of γ-aminobutyric acid and its easy and safe administration, prospective studies on pyridoxine supplementation in patients with eSE are needed.

Inhibition of human macrophage activation via pregnane neurosteroid interactions with toll-like receptors: Sex differences and structural requirements.

We recently discovered that (3α,5α)3-hydroxypregnan-20-one (allopregnanolone) inhibits pro-inflammatory toll-like receptor (TLR) activation and cytokine/chemokine production in mouse macrophage RAW264.7 cells. The present studies evaluate neurosteroid actions upon TLR activation in human macrophages from male and female healthy donors. Buffy coat leukocytes were obtained from donors at the New York Blood Center (http://nybloodcenter.org/), and peripheral blood mononuclear cells were isolated and cultured to achieve macrophage differentiation. TLR4 and TLR7 were activated by lipopolysaccharide (LPS) or imiquimod in the presence/absence of allopregnanolone or related neurosteroids and pro-inflammatory markers were detected by ELISA or western blotting. Cultured human monocyte-derived-macrophages exhibited typical morphology, a mixed immune profile of both inflammatory and anti-inflammatory markers, with no sex difference at baseline. Allopregnanolone inhibited TLR4 activation in male and female donors, preventing LPS-induced elevations of TNF-α, MCP-1, pCREB and pSTAT1. In contrast, 3α,5α-THDOC and SGE-516 inhibited the TLR4 pathway activation in female, but not male donors. Allopregnanolone completely inhibited TLR7 activation by imiquimod, blocking IL-1-ß, IL-6, pSTAT1 and pIRF7 elevations in females only. 3α,5α-THDOC and SGE-516 partially inhibited TLR7 activation, only in female donors. The results indicate that allopregnanolone inhibits TLR4 and TLR7 activation in cultured human macrophages resulting in diminished cytokine/chemokine production. Allopregnanolone inhibition of TLR4 activation was found in males and females, but inhibition of TLR7 signals exhibited specificity for female donors. 3α,5α-THDOC and SGE-516 inhibited TLR4 and TLR7 pathways only in females. These studies demonstrate anti-inflammatory effects of allopregnanolone in human macrophages for the first time and suggest that inhibition of pro-inflammatory cytokines/chemokines may contribute to its therapeutic actions.

Suppression of HIV-associated Macrophage Activation by a p75 Neurotrophin Receptor Ligand.

Previous studies indicated that nerve growth factor (NGF) and proNGF differentially regulate the phenotype of macrophages and microglia via actions at tropomyosin receptor kinase A (TrkA) and p75 neurotrophin receptors (p75NTR), respectively. The ability of HIV gp120 and virions to induce the secretion of factors toxic to neurons was suppressed by NGF and enhanced by proNGF, suggesting the potential for neurotrophin based "anti-inflammatory" interventions. To investigate the "anti-inflammatory" potential of the p75NTR ligand, LM11A-31, we treated cultured macrophages and microglia with HIV gp120 in the presence or absence of the ligand and evaluated the morphological phenotype, intrinsic calcium signaling, neurotoxic activity and proteins in the secretome. LM11A-31 at 10 nM was able to suppress the release of neurotoxic factors from both monocyte-derived macrophages (MDM) and microglia. The protective effects correlated with a shift in morphology and a unique secretory phenotype rich in growth factors that overrode the actions of HIV gp120. The protein pattern was generally consistent with anti-inflammatory, phagocytic and tissue remodeling functions. Although the toxic factor(s) and the source of the neuroprotection were not identified, the data indicated that an increased degradation of NGF induced by HIV gp120 was likely to contribute to neuronal vulnerability. Although substantial work is still needed to reveal the functions of many proteins in the mononuclear phagocyte secretome, such as growth and differentiation factors, the data clearly indicate that the ligand LM11A-31 has excellent therapeutic potential due to its ability to induce a more protective phenotype that restricts activation by HIV.

Tau seeds are subject to aberrant modifications resulting in distinct signatures.

The prion-like spread of tau pathology could underlie a spectrum of clinical syndromes including Alzheimer's disease (AD). Although evidence indicates that tau is transmissible, it is unclear how pathogenic tau seeds are processed in neurons. Here, we analyze fibrillar wild-type and disease-associated P301L tau seeds by using in vitro and neuronal assays. We show that P301L seeds are uniquely modified by post-translational modifications (PTMs) within the microtubule-binding region (MTBR). Although these modifications do not alter tau seed trafficking or localization, acetylated tau variants show accelerated tau aggregation, enhanced tau PTM priming, and prion-like templating. To explain the enhanced tau seed acetylation, we demonstrate that P301L seeds undergo auto-acetylation. Moreover, tau acts generally to inhibit HDAC6 deacetylase activity by preventing HDAC6 phosphorylation, leading to increased substrate acetylation. Our study highlights complex post-translational regulation of transmissible tau seeds and provides insight into the biological properties of tau strains in AD and other tauopathies.

GPR18 drives FAAH inhibition-induced neuroprotection against HIV-1 Tat-induced neurodegeneration.

Human immunodeficiency virus type 1 (HIV-1) is known to provoke microglial immune responses which likely play a paramount role in the development of chronic neuroinflammatory conditions and neuronal damage related to HIV-1 associated neurocognitive disorders (HAND). In particular, HIV-1 Tat protein is a proinflammatory neurotoxin which predisposes neurons to synaptodendritic injury. Drugs targeting the degradative enzymes of endogenous cannabinoids have shown promise in reducing inflammation with minimal side effects in rodent models. Considering that markers of neuroinflammation can predict the extent of neuronal injury in HAND patients, we evaluated the neurotoxic effect of HIV-1 Tat-exposed microglia following blockade of fatty acid amid hydrolyze (FAAH), a catabolic enzyme responsible for degradation of endocannabinoids, e.g. anandamide (AEA). In the present study, cultured murine microglia were incubated with Tat and/or a FAAH inhibitor (PF3845). After 24 h, cells were imaged for morphological analysis and microglial conditioned media (MCM) was collected. Frontal cortex neuron cultures (DIV 7-11) were then exposed to MCM, and neurotoxicity was assessed via live cell calcium imaging and staining of actin positive dendritic structures. Results demonstrate a strong attenuation of microglial responses to Tat by PF3845 pretreatment, which is indicated by 1) microglial changes in morphology to a less proinflammatory phenotype using fractal analysis, 2) a decrease in release of neurotoxic cytokines/chemokines (MCP-1/CCL2) and matrix metalloproteinases (MMPs; MMP-9) using ELISA/multiplex assays, and 3) enhanced production of endocannabinoids (AEA) using LC/MS/MS. Additionally, PF3845's effects on Tat-induced microglial-mediated neurotoxicity, decreased dysregulation of neuronal intracellular calcium and prevented the loss of actin-positive staining and punctate structure in frontal cortex neuron cultures. Interestingly, these observed neuroprotective effects appeared to be independent of cannabinoid receptor activity (CB1R & CB2R). We found that a purported GPR18 antagonist, CID-85469571, blocked the neuroprotective effects of PF3845 in all experiments. Collectively, these experiments increase understanding of the role of FAAH inhibition and Tat in mediating microglial neurotoxicity in the HAND condition.

Improved neurocognitive performance in FIV infected cats following treatment with the p75 neurotrophin receptor ligand LM11A-31.

HIV rapidly infects the central nervous system (CNS) and establishes a persistent viral reservoir within microglia, perivascular macrophages and astrocytes. Inefficient control of CNS viral replication by antiretroviral therapy results in chronic inflammation and progressive cognitive decline in up to 50% of infected individuals with no effective treatment options. Neurotrophin based therapies have excellent potential to stabilize and repair the nervous system. A novel non-peptide ligand, LM11A-31, that targets the p75 neurotrophin receptor (p75NTR) has been identified as a small bioavailable molecule capable of strong neuroprotection with minimal side effects. To evaluate the neuroprotective effects of LM11A-31 in a natural infection model, we treated cats chronically infected with feline immunodeficiency virus (FIV) with 13 mg/kg LM11A-31 twice daily over a period of 10 weeks and assessed effects on cognitive functions, open field behaviors, activity, sensory thresholds, plasma FIV, cerebrospinal fluid (CSF) FIV, peripheral blood mononuclear cell provirus, CD4 and CD8 cell counts and general physiology. Between 12 and 18 months post-inoculation, cats began to show signs of neural dysfunction in T maze testing and novel object recognition, which were prevented by LM11A-31 treatment. Anxiety-like behavior was reduced in the open field and no changes were seen in sensory thresholds. Systemic FIV titers were unaffected but treated cats exhibited a log drop in CSF FIV titers. No significant adverse effects were observed under all conditions. The data indicate that LM11A-31 is likely to be a potent adjunctive treatment for the control of neurodegeneration in HIV infected individuals.

Small molecule modulation of the p75 neurotrophin receptor suppresses age- and genotype-associated neurodegeneration in HIV gp120 transgenic mice.

The persistence of HIV in the central nervous system leads to cognitive deficits in up to 50% of people living with HIV even with systemic suppression by antiretroviral treatment. The interaction of chronic inflammation with age-associated degeneration places these individuals at increased risk of accelerated aging and other neurodegenerative diseases and no treatments are available that effectively halt these processes. The adverse effects of aging and inflammation may be mediated, in part, by an increase in the expression of the p75 neurotrophin receptor (p75NTR) which shifts the balance of neurotrophin signaling toward less protective pathways. To determine if modulation of p75NTR could modify the disease process, we treated HIV gp120 transgenic mice with a small molecule ligand designed to engage p75NTR and downregulate degenerative signaling. Daily treatment with 50 mg/kg LM11A-31 for 4 months suppressed age- and genotype-dependent activation of microglia, increased microtubule associated protein-2 (MAP-2), reduced dendritic varicosities and slowed the loss of parvalbumin immunoreactive neurons in the hippocampus. An age related accumulation of microtubule associated protein Tau was identified in the hippocampus in extracellular clusters that co-expressed p75NTR suggesting a link between Tau and p75NTR. Although the significance of the relationship between p75NTR and Tau is unclear, a decrease in Tau-1 immunoreactivity as gp120 mice entered old age (>16 months) suggests that the Tau may transition to more pathological modifications; a process blocked by LM11A-31. Overall, the effects of LM11A-31 are consistent with strong neuroprotective and anti-inflammatory actions that have significant therapeutic potential.

Escalating morphine dosing in HIV-1 Tat transgenic mice with sustained Tat exposure reveals an allostatic shift in neuroinflammatory regulation accompanied by increased neuroprotective non-endocannabinoid lipid signaling molecules and amino acids.

BACKGROUND: Human immunodeficiency virus type-1 (HIV-1) and opiates cause long-term inflammatory insult to the central nervous system (CNS) and worsen disease progression and HIV-1-related neuropathology. The combination of these proinflammatory factors reflects a devastating problem as opioids have high abuse liability and continue to be prescribed for certain patients experiencing HIV-1-related pain. METHODS: Here, we examined the impact of chronic (3-month) HIV-1 transactivator of transcription (Tat) exposure to short-term (8-day), escalating morphine in HIV-1 Tat transgenic mice that express the HIV-1 Tat protein in a GFAP promoter-regulated, doxycycline (DOX)-inducible manner. In addition to assessing morphine-induced tolerance in nociceptive responses organized at spinal (i.e., tail-flick) and supraspinal (i.e., hot-plate) levels, we evaluated neuroinflammation via positron emission tomography (PET) imaging using the [18F]-PBR111 ligand, immunohistochemistry, and cytokine analyses. Further, we examined endocannabinoid (eCB) levels, related non-eCB lipids, and amino acids via mass spectrometry.  RESULTS: Tat-expressing [Tat(+)] transgenic mice displayed antinociceptive tolerance in the tail withdrawal and hot-plate assays compared to control mice lacking Tat [Tat(-)]. This tolerance was accompanied by morphine-dependent increases in Iba-1 ± 3-nitrotryosine immunoreactive microglia, and alterations in pro- and anti-inflammatory cytokines, and chemokines in the spinal cord and striatum, while increases in neuroinflammation were absent by PET imaging of [18F]-PBR111 uptake. Tat and morphine exposure differentially affected eCB levels, non-eCB lipids, and specific amino acids in a region-dependent manner. In the striatum, non-eCB lipids were significantly increased by short-term, escalating morphine exposure, including peroxisome proliferator activator receptor alpha (PPAR-α) ligands N-oleoyl ethanolamide (OEA) and N-palmitoyl ethanolamide (PEA), as well as the amino acids phenylalanine and proline. In the spinal cord, Tat exposure increased amino acids leucine and valine, while morphine decreased levels of tyrosine and valine but did not affect eCBs or non-eCB lipids. CONCLUSION: Overall results demonstrate that 3 months of Tat exposure increased morphine tolerance and potentially innate immune tolerance evidenced by reductions in specific cytokines (e.g., IL-1α, IL-12p40) and microglial reactivity. In contrast, short-term, escalating morphine exposure acted as a secondary stressor revealing an allostatic shift in CNS baseline inflammatory responsiveness from sustained Tat exposure.

The Accumulation of Tau-Immunoreactive Hippocampal Granules and Corpora Amylacea Implicates Reactive Glia in Tau Pathogenesis during Aging.

The microtubule-associated tau protein forms pathological inclusions that accumulate in an age-dependent manner in tauopathies including Alzheimer's disease (AD). Since age is the major risk factor for AD, we examined endogenous tau species that evolve during aging in physiological and diseased conditions. In aged mouse brain, we found tau-immunoreactive clusters embedded within structures that are reminiscent of periodic acid-Schiff (PAS) granules. We showed that PAS granules harbor distinct tau species that are more prominent in 3xTg-AD mice. Epitope profiling revealed hypo-phosphorylated rather than hyper-phosphorylated tau commonly observed in tauopathies. High-resolution imaging and 3D reconstruction suggest a link between tau clusters, reactive astrocytes, and microglia, indicating that early tau accumulation may promote neuroinflammation during aging. Using postmortem human brain, we identified tau as a component of corpora amylacea (CA), age-related structures that are functionally analogous to PAS granules. Overall, our study supports neuroimmune dysfunction as a precipitating event in tau pathogenesis.

Modulation of the p75 neurotrophin receptor suppresses age-related basal forebrain cholinergic neuron degeneration.

Age-related degeneration of basal forebrain cholinergic neurons (BFCNs) is linked to cognitive impairment. The p75 neurotrophin receptor (p75NTR) has been proposed to mediate neuronal degeneration in aging. Therefore, we tested the hypothesis that modifying p75NTR function would prevent or reverse aging-related neuronal degeneration using LM11A-31, a small molecule p75NTR modulator that downregulates degenerative and upregulates trophic receptor-associated signaling. Morphological analysis in mice showed loss of BFCN area detectable by 18 months of age. Oral administration of LM11A-31 from age 15 to 18 months resulted in a dose-related preservation of BFCN area and one month of treatment from 17 to 18 months also preserved cell area. To evaluate reversal of established neuronal atrophy, animals were treated from 21 to 25 months of age. Treatment was associated with an increase of cell size to a mean area larger than that observed at 18 months, accompanied by increases in mean MS/VDB neurite length, as well as increased cholinergic fiber density and synaptophysin pre-synaptic marker levels in the hippocampus. These findings support the idea that modulation of p75NTR activity can prevent and potentially reverse age-associated BFCN degeneration. Moreover, this may be achieved therapeutically with orally bioavailable agents such as LM11A-31.

Enrichment Preferences of FIV-Infected and Uninfected Laboratory-Housed Cats.

Environmental enrichment is critical for alleviating stress in laboratory felines. However, there is a paucity of information about suitable enrichment for cats. This study aimed to determine preferred enrichment options of individually-housed, castrated male domestic short hair cats (Felis catus) used in a longitudinal study of the effects of chronic feline immunodeficiency virus (FIV) infection, and to determine if the FIV status of the cats affected enrichment preferences. Preference testing was performed with two types of grooming brushes, three different interactive play options, including a laser, ball, and petting interaction with a familiar investigator, and two types of toenail conditioning objects. We found that cats elected to be brushed, preferred social interaction and play with the laser to the ball, and preferred to scratch on an inclined-box toenail conditioning object compared to a horizontal, circular toenail conditioning object. There were individual preferences for enrichment opportunities. There were no differences in preferences between FIV-infected and sham-infected cats. These enrichment preferences may be used to advise laboratory animal facilities and researchers about how to best accommodate the behavioral needs of laboratory cats.

Feline Immunodeficiency Virus Neuropathogenesis: A Model for HIV-Induced CNS Inflammation and Neurodegeneration.

Feline Immunodeficiency virus (FIV), similar to its human analog human immunodeficiency virus (HIV), enters the central nervous system (CNS) soon after infection and establishes a protected viral reservoir. The ensuing inflammation and damage give rise to varying degrees of cognitive decline collectively known as HIV-associated neurocognitive disorders (HAND). Because of the similarities to HIV infection and disease, FIV has provided a useful model for both in vitro and in vivo studies of CNS infection, inflammation and pathology. This mini review summarizes insights gained from studies of early infection, immune cell trafficking, inflammation and the mechanisms of neuropathogenesis. Advances in our understanding of these processes have contributed to the development of therapeutic interventions designed to protect neurons and regulate inflammatory activity.

The Deacetylase HDAC6 Mediates Endogenous Neuritic Tau Pathology.

The initiating events that promote tau mislocalization and pathology in Alzheimer's disease (AD) are not well defined, partly because of the lack of endogenous models that recapitulate tau dysfunction. We exposed wild-type neurons to a neuroinflammatory trigger and examined the effect on endogenous tau. We found that tau re-localized and accumulated within pathological neuritic foci, or beads, comprised of mostly hypo-phosphorylated, acetylated, and oligomeric tau. These structures were detected in aged wild-type mice and were enhanced in response to neuroinflammation in vivo, highlighting a previously undescribed endogenous age-related tau pathology. Strikingly, deletion or inhibition of the cytoplasmic shuttling factor HDAC6 suppressed neuritic tau bead formation in neurons and mice. Using mass spectrometry-based profiling, we identified a single neuroinflammatory factor, the metalloproteinase MMP-9, as a mediator of neuritic tau beading. Thus, our study uncovers a link between neuroinflammation and neuritic tau beading as a potential early-stage pathogenic mechanism in AD.

Novel p75 neurotrophin receptor ligand stabilizes neuronal calcium, preserves mitochondrial movement and protects against HIV associated neuropathogenesis.

Human immunodeficiency virus (HIV) rapidly penetrates into the brain and establishes a persistent infection of macrophages/microglia. Activation of these cells by HIV results in the secretion of soluble factors that destabilize neuronal calcium homeostasis, encourage oxidative stress and result in neural damage. This damage is thought to underlie the cognitive-motor dysfunction that develops in many HIV-infected patients. Studies have suggested that neurotrophins may protect neurons from the toxic effects of HIV-associated proteins. To better understand the pathogenic mechanisms and the neuroprotective potential of neurotrophin ligands, we evaluated neuronal damage, calcium homeostasis and mitochondrial functions after exposure of cultured rat neurons directly to HIV gp120 or to conditioned medium from human monocyte-derived macrophages treated with gp120. We then assessed the ability of a new non-peptide p75 neurotrophin receptor ligand, LM11A-31, to stabilize calcium homeostasis and prevent the development of pathology. Each toxic challenge resulted in a delayed accumulation of intracellular calcium coupled to a decrease in the rate of calcium clearance from the cell. The delayed calcium accumulation correlated with the development of focal dendritic swellings (beading), cytoskeletal damage and impaired movement of mitochondria. Addition of LM11A-31 to the cultures at nanomolar concentrations eliminated cell death, significantly reduced the pathology, suppressed the delayed accumulation of calcium and restored mitochondrial movements. The potent neuroprotection and the stabilization of calcium homeostasis indicate that LM11A-31 may have excellent potential for the treatment of HIV-associated neurodegeneration.

Opposing Effects of NGF and proNGF on HIV Induced Macrophage Activation.

Macrophage and microglial activation by HIV in the central nervous system (CNS) triggers the secretion of soluble factors which damage neurons. Therapeutic approaches designed to restore cognitive function by suppressing this inflammatory activity have not yet been successful. Recent studies have indicated that the phenotype of macrophages is differentially controlled by the mature and pro form of nerve growth factor. These cells therefore may be highly responsive to the imbalance in pro versus mature neurotrophins often associated with neurodegenerative diseases. In this study we evaluated the interactions between neurotrophins and HIV induced macrophage activation. HIV stimulation of macrophages induced a neurotoxic phenotype characterized by the expression of podosomes, suppression of calcium spiking and increased neurotoxin production. The secretome of the activated macrophages revealed a bias toward anti-angiogenic like activity and increased secretion of MMP-9. Co-stimulation with NGF and HIV suppressed neurotoxin secretion, increased calcium spiking, suppressed podosome expression and reversed 86% of the proteins secreted in response to HIV, including MMP-9 and many growth factors. In contrast, co-stimulation of macrophages with proNGF not only failed to reverse the effects of HIV but increased the neurotoxic phenotype. These differential effects of proNGF and NGF on HIV activation provide a potential novel therapeutic avenue for controlling macrophage activation in response to HIV.