SERPIN-Derived Small Peptide (SP16) as a Potential Therapeutic Agent against HIV-Induced Inflammatory Molecules and Viral Replication in Cells of the Central Nervous System.

Despite the success of combined antiretroviral therapy (cART) increasing the survival rate in human immunodeficiency virus (HIV) patients, low levels of viremia persist in the brain of patients leading to glia (microglia and astrocytes)-induced neuroinflammation and consequently, the reactivation of HIV and neuronal injury. Here, we tested the therapeutic efficacy of a Low-Density Lipoprotein Receptor-Related Protein 1 (LRP-1) agonistic small peptide drug (SP16) in attenuating HIV replication and the secretion of inflammatory molecules in brain reservoirs. SP16 was developed by Serpin Pharma and is derived from the pentapeptide sequence of the serine protease inhibitor alpha-1-antitrypsin (A1AT). The SP16 peptide sequence was subsequently modified to improve the stability, bioavailability, efficacy, and binding to LRP-1; a scavenger regulatory receptor that internalizes ligands to induce anti-viral, anti-inflammatory, and pro-survival signals. Using glial cells infected with HIV, we showed that: (i) SP16 attenuated viral-induced secretion of pro-inflammatory molecules; and (ii) SP16 attenuated viral replication. Using an artificial 3D blood-brain barrier (BBB) system, we showed that: (i) SP16 was transported across the BBB; and (ii) restored the permeability of the BBB compromised by HIV. Mechanistically, we showed that SP16 interaction with LRP-1 and binding lead to: (i) down-regulation in the expression levels of nuclear factor-kappa beta (NF-κB); and (ii) up-regulation in the expression levels of Akt. Using an in vivo mouse model, we showed that SP16 was transported across the BBB after intranasal delivery, while animals infected with EcoHIV undergo a reduction in (i) viral replication and (ii) viral secreted inflammatory molecules, after exposure to SP16 and antiretrovirals. Overall, these studies confirm a therapeutic response of SP16 against HIV-associated inflammatory effects in the brain.

Implication of the Autophagy-Related Protein Beclin1 in the Regulation of EcoHIV Replication and Inflammatory Responses.

The protein Beclin1 (BECN1, a mammalian homologue of ATG6 in yeast) plays an important role in the initiation and the normal process of autophagy in cells. Moreover, we and others have shown that Beclin1 plays an important role in viral replication and the innate immune signaling pathways. We previously used the cationic polymer polyethyleneimine (PEI) conjugated to mannose (Man) as a non-viral tool for the delivery of a small interfering (si) Beclin1-PEI-Man nanoplex, which specifically targets mannose receptor-expressing glia (microglia and astrocytes) in the brain when administered intranasally to conventional mice. To expand our previous reports, first we used C57BL/6J mice infected with EcoHIV and exposed them to combined antiretroviral therapy (cART). We show that EcoHIV enters the mouse brain, while intranasal delivery of the nanocomplex significantly reduces the secretion of HIV-induced inflammatory molecules and downregulates the expression of the transcription factor nuclear factor (NF)-kB. Since a spectrum of neurocognitive and motor problems can develop in people living with HIV (PLWH) despite suppressive antiretroviral therapy, we subsequently measured the role of Beclin1 in locomotor activities using EcoHIV-infected BECN1 knockout mice exposed to cART. Viral replication and cytokine secretion were reduced in the postmortem brains recovered from EcoHIV-infected Becn1+/- mice when compared to EcoHIV-infected Becn1+/+ mice, although the impairment in locomotor activities based on muscle strength were comparable. This further highlights the importance of Beclin1 in the regulation of HIV replication and in viral-induced cytokine secretion but not in HIV-induced locomotor impairments. Moreover, the cause of HIV-induced locomotor impairments remains speculative, as we show that this may not be entirely due to viral load and/or HIV-induced inflammatory cytokines.

Targeting Beclin1 as an Adjunctive Therapy against HIV Using Mannosylated Polyethylenimine Nanoparticles.

Using nanoparticle-based RNA interference (RNAi), we have previously shown that silencing the host autophagic protein, Beclin1, in HIV-infected human microglia and astrocytes restricts HIV replication and its viral-associated inflammatory responses. Here, we confirmed the efficacy of Beclin1 small interfering RNA (siBeclin1) as an adjunctive antiviral and anti-inflammatory therapy in myeloid human microglia and primary human astrocytes infected with HIV, both with and without exposure to combined antiretroviral (cART) drugs. To specifically target human microglia and human astrocytes, we used a nanoparticle (NP) comprised of linear cationic polyethylenimine (PEI) conjugated with mannose (Man) and encapsulated with siBeclin1. The target specificity of the PEI-Man NP was confirmed in vitro using human neuronal and glial cells transfected with the NP encapsulated with fluorescein isothiocyanate (FITC). PEI-Man-siBeclin1 NPs were intranasally delivered to healthy C57BL/6 mice in order to report the biodistribution of siBeclin1 in different areas of the brain, measured using stem-loop RT-PCR. Postmortem brains recovered at 1-48 h post-treatment with the PEI-Man-siRNA NP showed no significant changes in the secretion of the chemokines regulated on activation, normal T cell expressed and secreted (RANTES) and monocyte chemotactic protein-1 (MCP-1) and showed significant decreases in the secretion of the cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) when compared to phosphate-buffered saline (PBS)-treated brains. Nissl staining showed minimal differences between the neuronal structures when compared to PBS-treated brains, which correlated with no adverse behavioral affects. To confirm the brain and peripheral organ distribution of PEI-siBeclin1 in living mice, we used the In vivo Imaging System (IVIS) and demonstrated a significant brain accumulation of siBeclin1 through intranasal administration.

Impact of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in the Nervous System: Implications of COVID-19 in Neurodegeneration.

Coronavirus Disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2), began in December 2019, in Wuhan, China and was promptly declared as a pandemic by the World Health Organization (WHO). As an acute respiratory disease, COVID-19 uses the angiotensin-converting enzyme 2 (ACE2) receptor, which is the same receptor used by its predecessor, SARS-CoV, to enter and spread through the respiratory tract. Common symptoms of COVID-19 include fever, cough, fatigue and in a small population of patients, SARS-CoV-2 can cause several neurological symptoms. Neurological malaise may include severe manifestations, such as acute cerebrovascular disease and meningitis/encephalitis. Although there is evidence showing that coronaviruses can invade the central nervous system (CNS), studies are needed to address the invasion of SARS-CoV-2 in the CNS and to decipher the underlying neurotropic mechanisms used by SARS-CoV-2. This review summarizes current reports on the neurological manifestations of COVID-19 and addresses potential routes used by SARS-CoV-2 to invade the CNS.