Knockout of angiotensin converting enzyme-2 receptor leads to morphological aberrations in rodent olfactory centers and dysfunctions associated with sense of smell.

Neuronal morphological characterization and behavioral phenotyping in mouse models help dissecting neural mechanisms of brain disorders. Olfactory dysfunctions and other cognitive problems were widely reported in asymptomatic carriers and symptomatic patients infected with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). This led us to generate the knockout mouse model for Angiotensin Converting Enzyme-2 (ACE2) receptor, one of the molecular factors mediating SARS-CoV-2 entry to the central nervous system, using CRISPR-Cas9 based genome editing tools. ACE2 receptors and Transmembrane Serine Protease-2 (TMPRSS2) are widely expressed in the supporting (sustentacular) cells of human and rodent olfactory epithelium, however, not in the olfactory sensory neurons (OSNs). Hence, acute inflammation induced changes due to viral infection in the olfactory epithelium may explain transient changes in olfactory detectabilities. As ACE2 receptors are expressed in different olfactory centers and higher brain areas, we studied the morphological changes in the olfactory epithelium (OE) and olfactory bulb (OB) of ACE2 KO mice in comparison with wild type animals. Our results showed reduced thickness of OSN layer in the OE, and a decrease in cross-sectional area of glomeruli in the OB. Aberrations in the olfactory circuits were revealed by lowered immunoreactivity toward microtubule associated protein 2 (MAP2) in the glomerular layer of ACE2 KO mice. Further, to understand if these morphological alterations lead to compromised sensory and cognitive abilities, we performed an array of behavioral assays probing their olfactory subsystems' performances. ACE2 KO mice exhibited slower learning of odor discriminations at the threshold levels and novel odor identification impairments. Further, ACE2 KO mice failed to memorize the pheromonal locations while trained on a multimodal task implying the aberrations of neural circuits involved in higher cognitive functions. Our results thus provide the morphological basis for the sensory and cognitive disabilities caused by the deletion of ACE2 receptors and offer a potential experimental approach to study the neural circuit mechanisms of cognitive impairments observed in long COVID.

Multimodal learning of pheromone locations.

Memorizing pheromonal locations is critical for many mammalian species as it involves finding mates and avoiding competitors. In rodents, pheromonal information is perceived by the main and accessory olfactory systems. However, the role of somatosensation in context-dependent learning and memorizing of pheromone locations remains unexplored. We addressed this problem by training female mice on a multimodal task to locate pheromones by sampling volatiles emanating from male urine through the orifices of varying dimensions or shapes that are sensed by their vibrissae. In this novel pheromone location assay, female mice' preference toward male urine scent decayed over time when they were permitted to explore pheromones vs neutral stimuli, water. On training them for the associations involving olfactory and whisker systems, it was established that they were able to memorize the location of opposite sex pheromones, when tested 15 days later. This memory was not formed either when the somatosensory inputs through whisker pad were blocked or when the pheromonal cues were replaced with that of same sex. The association between olfactory and somatosensory systems was further confirmed by the enhanced expression of the activity-regulated cytoskeleton protein. Furthermore, the activation of main olfactory bulb circuitry by pheromone volatiles did not cause any modulation in learning and memorizing non-pheromonal volatiles. Our study thus provides the evidence for associations formed between different sensory modalities facilitating the long-term memory formation relevant to social and reproductive behaviors.

Promoter polymorphism MMP-1 (-1607 2G/1G) and MMP-3 (-1612 5A/6A) in development of HAND and modulation of pathogenesis of HAND.

The pathogenesis of HIV-associated neurocognitive disorder (HAND) is modulated by host genetic susceptibility factors such as Matrix metalloproteinases (MMPs). Promoter polymorphism of MMP-1 and MMP-3 may modify the expression of the gene. Hence, we evaluated the association of MMP-1-16072G/1G and MMP-3-1612 5A/6A polymorphisms with development of HAND and the modulation of pathogenesis of HAND. We enrolled a total of 180 individuals, 50 HIVinfected individuals with HAND, 130 without HAND, and 150 healthy controls. Polymorphism of MMP-1 and MMP-3 were genotyped by PCR-RFLP. MMP-1-1607 2G1G, -16071G/2G-1G/1G genotypes and -1607 1G allele were associated with the development of HAND (OR = 1.64, P = 0.05; OR = 1.45, P = 0.04; OR = 1.69, P = 0.05). MMP-1- 16071G1G, MMP-3-16125A5A genotypes increased the risk for the development of HAND (OR = 1.78, P = 0.25; OR = 2.39, P = 0.13). MMP-3-1612 5A5A, -1612 6A/5A-5A/5A genotypes and -1612 5A allele were associated with the reduced risk of HAND (OR = 0.40, P = 0.05; OR = 0.53, P = 0.04; OR = 0.40, P = 0.01). Haplotype 5A1G increased the risk of development of HAND (OR = 1.93, P = 0.05). As observed in advanced HIV disease stage, MMP-1-1607 1G1G genotype enhance the risk for advancement of HIV disease (OR = 1.69, P = 0.89). MMP-3-1612 6A5A genotype showed higher risk for development of HAND in alcohol users (0R = 1.65, P = 0.44). MMP-1 genotype may have an influence on development of HAND whereas MMP3-1612 5A5A genotype may reduce risk for pathogenesis of HAND.

Genetic variation of MMP-2(-735 C>T) and MMP-9(-1562 C>T) gene in risk of development of HAND and severity of HAND.

BACKGROUND: Astrocytes are susceptible to HIV-1 infection. Neurocognitive dysfunction has also been associated with the toxicity of certain antiretroviral drugs. HIV-1 induced neurological toxicity has been associated with deficiency of matrix metalloproteinases. Therefore, we evaluated the association of MMP-2(-735C > T) and MMP-9(-1562C > T) polymorphisms with respect to the susceptibility of developing HIV-associated neurocognitive disorders (HAND) and its severity. METHODS: We enrolled 50 HIV-infected individuals with HAND, 130 without HAND and 150 unrelated healthy controls. Polymorphism for MMP-2-735C > T and MMP-9-1562C > T genes was genotyped by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: Individuals with the MMP-2 -735 CT genotype and -735 T allele were at higher risk of developing HAND [odds ratio (OR) = 5.27, 95% confidence interval (CI) = 1.30-21.35, p = 0.02 and OR = 2.27, 95% CI = 1.57-3.27, p = 0.0001 respectively]. The MMP-2 -735 CT genotype and -735 T allele of MMP-2 were associated with a reduced likelihood of severe HAND (OR =0.32, 95% CI = 0.15-0.66, p = 0.002 and OR = 0.32, 95% CI = 0.14-0.71, p = 0.005). When evaluating gene-gene interaction models, the combined genotype MMP-2-735TT + MMP-9-1562CC and MMP-2-735CT + MMP-9-1562CT was associated with the risk of developing HAND (OR = 4.84, p = 0.0001, OR = 1.81, p = 0.03). However, individuals with the combined genotype of MMP-2-735TT + MMP-9-1562CC were found to be protective for severe HAND (OR = 0.30, 95% CI = 0.13-0.67, p = 0.003). CONCLUSIONS: Individuals with the MMP-2 -735CT genotype, -735 T allele and combined genotype MMP-2 -735TT + MMP-9 -1562CC had an enhanced risk of developing HAND. Those with the MMP-2 -735 CT genotype, -735 T allele and combined genotype of MMP-2-735TT + MMP-9-1562CC were suggested to have protection from developing severe HAND.

APOBEC3B deletion impacts on susceptibility to acquire HIV-1 and its advancement among individuals in western India.

APOBEC3B deletion polymorphism has been associated with risk of HIV-1 acquisition and its progression. Therefore, we aimed to investigate the association of APOBEC3B ins/del polymorphism with risk of acquisition of HIV-1 and its progression. In the present case-control study, we enrolled a total of 150 HIV-infected individuals and 150 healthy controls. Polymorphism for APOBEC3B gene was genotyped by PCR. APOBEC3B ID, DD genotypes, and D allele were associated with higher risk of acquisition of HIV-1 (p = 0.004, OR = 4.96; p = 0.03, OR = 3.55; and p = 0.004; OR = 1.60). The individuals with ID genotypes and combined genotype ID+DD of APOBEC3B in the presence of tobacco and alcohol showed the higher risk of advancement of HIV disease; however, risk could not reach statistical significance (OR = 1.14, 95% CI: 0.59-2.18; OR = 1.33, 95% CI: 0.83-2.15 and OR = 1.44, 95% CI: 0.77-2.69; OR = 1.50, 95% CI: 0.94-2.40). Individuals in advanced HIV disease stage and ID genotype and combined genotype ID + DD of APOBEC3B were more likely to be associated with advanced HIV disease stage but risk could not reach significant (OR = 1.50, 95% CI: 0.94-2.40; OR = 1.27, 95% CI: 0.88-1.84). Individuals with ID and DD genotype of APOBEC3B had influence on susceptibility to acquisition of HIV-1. This suggests that APOBEC3B deletion may attenuate innate cellular immunity against HIV-1 and thus confer the host persistence for HIV infection.

Impact of variants of MMP-7(-181A>G) gene in susceptibility to the development of HIV-associated neurocognitive disorder and its severity.

The HIV-1-induced neurological toxicity has been associated with the deficiency of matrix metalloproteinases. Tat protein of HIV up regulates MMP-7 release and activation, leading to neurotoxicity. The SNP -181A>G of MMP-7 is known to have functional effects on its promoter activity. Therefore, we aimed to evaluate the association of variants of MMP-7 -181A>G gene in HIV-associated neurocognitive disorder (HAND). In the present case-control study, we recruited 50 HIV-infected individuals with HAND, 130 HIV-infected individuals without HAND and 150 unrelated healthy individuals. Polymorphism for MMP-7 -181A>G gene was genotyped by PCR-RFLP method. Frequency of -181GG and G allele of MMP-7 did not differ significantly between patients with HAND and without HAND (8.0% vs 13.1%, p = 0.22 and 31% vs 38.1%, p = 0.21). Individuals with -181 AG, -181GG genotype, and G allele of MMP-7 were found to have reduced the risk of development of HAND but not significant (50.0% vs 51.9%, p = 0.09, OR = 0.54; 13.1% vs 19.0%, p = 0.33, OR = 0.71 and 38.1% vs 44.9%, p = 0.09, OR = 0.75). Individuals in early HIV disease stage having -181AG genotype and -181AG + GG combined genotype of MMP-7 were not associated with the development of HAND (OR = 1.27, p = 0.25 and OR = 1.25, p = 0.17). Tobacco and alcohol consumption among individuals with any genotype of MMP-7 was not associated with the risk of development of HAND. In conclusion, individuals with -181GG genotype and G allele had no impact on susceptibility to the development of HAND and its severity.