Genomic benchmarking studies reveal variations of the polyubiquitination domain of the PSD 95 protein in Homo neanderthalensis and other primates of the Hominidae family : Possible implications in cognitive functions ?

Modern humans' unique cognitive abilities regarding Neanderthals and other primate's lineages are frequently attributed to the differences in brain size development and evolution. However, recent studies have established the critical role of genomic and genetic benchmarking in analyzing the cognitive evolution between modern humans and primates, focused mainly on searching for involved genes in neurogenesis. PSD95 protein (named PSD95p) has a key role in modulating synaptic plasticity, learning, and memory skills. Thus, the present study aimed to determine the possible variations of the PSD95 gene between modern humans, Neanderthals, and other hominid primate species using bioinformatics tools. The results showed 14 polymorphisms compared with the contemporary human PSD95 gene, of which 13 were silent mutations, and only one was a non-silent mutation at the nucleotide position 281. Despite polymorphisms found at the nucleotide sequences, the PSD95p of humans and chimpanzees are 100% identical. Likewise, the gorilla and orangutan PSD95p are 100% identical, although a 103-amino acid deletion characterizes them at the N-terminal end (1-103), suggesting that it behaves like a non-functional protein. Interestingly, the single nucleotide polymorphism (SNP) found at position 281 in the Neanderthal PSD95 gene leads to a change of the E94 to valine V94 in the polyubiquitination domain (PEST) and variation in the three-dimensional structure of PSD95 protein. We prompt that this structural change in the PEST domain could induce a loss of PSD95p function and, therefore, an alteration in synaptic plasticity forms such as long-term potentiation (LTP) and long-term depression (LTD). These findings open a possible hypothesis supporting the idea that humans' cognitive evolution after separating our last common ancestor with Neanderthals lineage could have been accompanied by discrete changes in the PSD95p polyubiquitination domain.


Introduction
Since our last common ancestor's divergence with Neanderthals, the early humans' evolution process, between 430,000 to 765,000 years ago [1][2][3][4] , and with chimpanzees, between 4 to 7 million years ago 5,6 , has become a subject of great controversy. One of the most relevant issues within this matter is precisely the interbreeding that humans and Neanderthals could have after migrated from Sub-Saharan Africa in two events. The first time carried out 100,000 years ago 4 and the second time 50,000 years ago 3,[7][8][9][10] . Based on studies of fossil remains, it has been reported that the second interbreeding event allowed the introgression of around 6-9% of the Neanderthal genome into the early human genome 4,7 . However, due to positive selection processes, a recent Asian and European genomes analysis has shown that the percentage of genetic segments introgressed from Neanderthal to human fell 1-3% 3,7,11,12 .
The persistent Neanderthal genes in the genome of modern non-African humans are mainly those involved in phenotypic traits, directly influenced by the environment and other genes involved in both cognitive and skeletal development 8,9 . Consequently, these genes have been linked to the expansion of modern humans throughout Europe, which subsequently led to the disappearance of Neanderthals 30,000 years ago 8,13 . Nevertheless, it is evident that beyond a disappearance, Neanderthals were diluted in the early human genome.
On the other hand, despite the evolutionary processes in time and space between modern humans and chimpanzees, unlike Neanderthals, it has been found that their genome is 98.7% identical 5,6 . Despite their high identity percentage, likewise other hominids, chimpanzees also differ from modern humans in many morphological, behavioral, and cognitive characteristics 5 .
Modern humans' behavior and unique cognitive abilities concerning Neanderthals and other hominid primates have been attributed to the brain's evolution process 5,[14][15][16][17][18] . Several studies have suggested that the lower development of the brain's cranial capacity and organization could be the possible cause of the Neanderthal extinction 14,[18][19][20] . Based on these facts, it has been reported that the Neanderthal adaptive system was unable to survive the competitive environment with modern humans [20][21][22] , therefore, emphasizing that modern humans had a more remarkable cognitive ability to develop adaptive strategies 4,7 . However, a report described that the Neanderthals and modern humans had similar brain sizes, advocating that it is wrong to infer differentiated cognitive development between these two species based on archaeological data analysis 23 . Recent studies reveal the importance of molecular biology and bioinformatics tools for the cognitive development investigation between modern humans and their common ancestors, which are focused mainly on the search of involved genes in neurogenesis, a mechanism associated with learning and memory developments 6,18,24 . Therefore, it has been suggested that the evolutionary development of the brain is a direct cause of a more significant generation of neural networks, hence the rounded form that has been attributed to the modern human brain compared with the elongated shape of the Neanderthal brain 24 .
Likewise, it has been found that the regulation of genes involved in the migration and detrital development of neurons has led to a dissimilar functional development between the neurons of humans, chimpanzees, and bonobo 18 . In-silico analysis done in this work are not enough to confirm that the polymorphism of the PSD95 can be associated with neural networks. However, the results open a window to the scientific community because they allow knowing and infer a possible cause in the cognitive development differences between Neanderthals and modern humans and other primates.
It is known that the learning process through memory lies in the organization of neurons and their interactions 25 . Memories are stored in a neuronal circuit by synapse mechanisms and specific neurons 26 . Synaptic plasticity or dynamic changes of neuronal connections change the neuron's chemical composition and its capacity to communicate with its neighbors. Also, that allows the generation of new functional neurons (neurogenesis), contributing even more to plasticity 27 . The me-chanisms involved in synaptic plasticity changes are long-term potentiation (LTP) and long-term depression (LTD), characterized by an increase and weakening of the synaptic force, respectively. Therefore, the favoring of LTP and the development of memory and learning have been associated with an increased expression of the PSD95 gene, which encodes a protein of postsynaptic density 95 (PSD95p) characteristic of an electrodense region called postsynaptic density 28,29 .
PSD95p is a specialized scaffold protein composed of several multiple protein interaction domains that include: i) a polyubiquitination domain (PEST), ii) three PDZ domains, iii) an SH3 domain, and iv) a guanylate kinase (GK)-like domain [30][31][32] . PSD-95p is the best-studied protein of the guanylate kinase-associated membrane family (MAGUK) 30 . Within this family, there are other scaffolding proteins such as SAP102, SAP97, and PSD93 32 . PSD95p with PSD93p can interact in postsynaptic sites to form a multimeric scaffold for grouping associated receptors, ion channels, and signaling proteins 30,33,34 . As shown in Figure. 1, PSD95p is the anchoring axis of a complex of postsynaptic proteins, ion channels, and NMDA receptors (N-methyl-D-aspartate) and AMPA (α-amino-3-hydroxy-5- methyl-4-acid isoxazole propionic) type. The presence of the NMDA and AMPA proteins is essential in the synaptic plasticity and cognitive functions development. However, their correct function will depend directly on the presence of PSD95p. For this reason, this study was only focused on the function of PSD95p, which plays an essential role in synaptic plasticity during LTP by allowing better localization of AMPA receptors in the synaptic membrane; thus, inducing an increase in the synaptic connections' strength between neighboring neurons [33][34][35][36][37] .
Experiments performed in mice have shown that mutations in the gene that encodes the PSD95 protein lead to a deficit in learning and memory abilities, abnormal social behavior, repetitive behaviors, increased stress, anxiety-related responses, and different motor alterations 34,38 . In the case of mutations and erroneous genetic variants in the ARCp-PS-D95p complex, it has been found that there may be a high susceptibility to schizophrenia, autism disorders, intellectual disabilities, epileptic mutations, and abnormal variants in intelligence 33,39,40 . In future works, mutation analysis in the other proteins involved with cognitive development, such as ARCp, AMPA, and NMDA receptors, among others, will be elucidated.
In this research, we hypothesized that if in an evolutionary past, humans acquired specific variations in the nucleotide sequence of the PSD95 gene, then the structure and function of PSD95p, as well as the development of cognitive abilities, could have been improved compared to Neanderthals and other primates of the Hominidae family.
(Neanderthal), Pan troglodytes (Chimpanzee), Pan paniscus (Bonobo), Gorilla gorilla (Gorilla) and Pongo abelii (Orangutan). The PSD95 nucleotide (without introns) and PSD95p amino acid sequences of human, Chimpanzee, bonobo, gorilla, and orangutan were downloaded from the Ensembl Database 41 and GenBank database 42 . The Neanderthal's nucleotide and amino acid sequences were downloaded from the Neanderthal Genome database 43 , as shown in Tables 1 and 2.
Due to the present study's complexity, only one sequence was selected per Hominidae primate's species studied. However, in the case of Neanderthals, there is only one access to a sequenced genome in the Neanderthals Genome database.
In spite that this approach only uses one sequence by a specimen to make the comparison between species regarding PSD95 genes and proteins, it is useful to release scientific questions and hypothesis and to planning strong in vivo and in vitro experiments allowing to determine the relationships between variations of the polyubiquitination domain of the PSD95 protein and its possible implications in cognitive functions in Homo neanderthalensis and other primates of the Hominidae family, including modern human adequately.

Prediction of 3D protein structures
Prediction of the tertiary structure of human PSD5p was performed with Protein Homology/analogy Recognition Engine V 2.0 (PHYRE2) server 45 , which also allowed us to predict the 3D structure domains have not been crystallized previously. Prediction of the PSD5p 3D structures of Neanderthal, Chim- Table 1. General characteristics of the studied Hominidae primates PSD95 genes. Table 2. General characteristics of the studied Hominidae primates PSD95 proteins. The multiple sequence alignments were performed with the MUSCLE server directly linked to the Molecular Evolutionary Genetics Analysis software, MEGA X 10.0.5 44

Materials and methods
Obtaining PSD95 sequences and multiple alignments The primates selected for the present study were chosen based on the species belonging to the Hominidae clade, specifically: Homo sapiens (Human), Homo neanderthalensis panzee, bonobo, gorilla, and orangutan was performed by SWISS-MODEL server 46 previous model generated for humans as a template. Finally, the models obtained were analyzed using PyMOL 2.3 software 47 .

Phylogenetic trees
Two phylogenetic trees were built using nucleotide and amino acid sequences of the PSD95 gene and the PSD95p. The evolutionary history was performed using MEGA X 48 , the maximum likelihood method. Additionally, the Tamura-Nei model was used 49 for nucleotides and the model Le_Gascuel_2008 50 for amino acids. To increase the reliability of the analyzed taxa's consensus trees, a bootstrap corresponding to 2500 replicates was established 51 .
To decrease the construction complexity of the consensus tree that presents an optimal topology, the heuristic search method was used. For this, an initial tree was automatically obtained, applying the algorithms of Neighbor-Join and BioNJ to a matrix of distances by pairs estimated using the Maximum Composition Probability (MCL) approach, for the nucleotide sequences. The heuristic search's initial tree was obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using a JTT model. The analysis involved 6 nucleotide sequences and 6 amino acid sequences.

Results
Polymorphisms are found in the PEST domain of the PSD95 gene in Neanderthal and other primates of the Hominidae family Despite the high percentage of identity (Table 1), the results obtained showed 14 polymorphisms along the PSD95 gene ( Figure.   Interestingly, as is shown in Figure 2B, the alignments of amino acid sequences showed that, from the 14 polymorphisms shown above, 13 correspond to silent mutations. Only one mutation is a non-silent type, which was found in the Neanderthal PSD95 gene. This single nucleotide polymorphism (SNP), based on substitution of adenine by thymine at position 281, led to a change of the glutamate residue at position 94 (E94R) by a valine (V94R) in the Neanderthal PSD95 protein.
Similarly, the deletion of the 9 nucleotides in bonobo led to a modification of 3 amino acids (E94R-L95R-Q96R), and the deletion of 309 nucleotides in gorilla and orangutan led to a deletion of 103 amino acids at the N-terminal end of the protein (position 1-103). On the other hand, the results indicate that, despite the polymorphisms found in the nucleotide sequences, the PSD95p protein from humans and chimpanzees are 100% identical. Likewise, gorilla and orangutan proteins do not show specific amino acid changes. An important fact is that overall substitutions and deletions from species studied were found in the PEST domain of PSD95p, as shown in Figure 2C.
The evolutionary history based on the PSD95p protein indicates that modern humans have a greater kinship with chimpanzees than with Neanderthals.
Using the maximum likelihood method, the evolutionary lineage concerning the PSD95 gene shows us the three clades' formation; the first one groups includes human, Neanderthal, and gorilla. In this clade, we can observe that humans have a greater kinship with Neanderthals than with gorillas ( Figure.  3A). The second clade includes Chimpanzee and bonobo, and the third one only consists of the orangutan.
Regarding the PSD95p protein, three clades are obtained ( Figure. 3B). However, because the polymorphisms found were non-silent mutations, except for Neanderthal, differences were observed in the first two clades described above for the phylogenetic tree regarding the species' PSD95 genes studied. Thus, a first clade was observed for humans and chimpanzees, a second clade for Neanderthals, bonobo, and gorilla, where Neanderthal has a greater kinship with bonobo. Finally, no differences were observed in the third clade formed by the orangutan.

Discussion
Proteins can fold in a specific way depending on the amino acids that constitute them until forming three-dimensional structures. Consequently, the function of a protein is directly related to its structure. Based on the results based on PSD95p sequences, we have shown that chimpanzees have a higher percentage of identity with modern humans. However, studies have shown that the cognitive differences between these two species are not governed solely by structural changes in gene products as proteins and gene expression levels are involved in the different cognitive processes.
On the other hand, due to the SNP found in the Neanderthal PSD95 gene, a change of glutamate © by valine (V) is generated, leading to a modification in the 3D conformational of the protein, as shown in Figure. 4A-B. Thus, we believe that the function of the PSD95 protein from Neanderthal was possibly affected, with a concomitant reduction in cognitive function compared with modern humans.
Likewise, as a consequence of the SNP found in the Neanderthal PSD95 gene, a change of glutamate © by valine (V) is generated, leading to a modification in the 3D conformational of the protein and therefore in its function ( Figure. 4).
According to previous studies, a change of the glutamate amino acid within the PEST domain may lose its function. The synaptic strength depends on the AMPA and NMDA type receptors, regulated in part by the PSD95 protein, which seems depleted 52,53 . Then, based on the conservation of the E94R-L95R-Q96R motif ( Figure. 5), it could be thought that the modification in the E94R residue in Neanderthal could have induced a loss of function of the PSD95p protein. Likewise, the signal peptide's de-palmitoylation at the N-terminal end of the protein can weaken synaptic strength and lead to an LTD. This sequence of about 24 amino acids, found upstream of the PEST domain when undergoing a palmitoylation process in cysteine, serine, or threonine residues, allows PSD95p to be coupled postsynaptic membrane for subsequent anchoring of receptors and other signaling and adhesion proteins 36,54,55 . Therefore, based on the mentioned above, it could be inferred that the deletion of the N-terminal end of 103 amino acids in the gorilla and orangutan protein may be, among other elements, a probable cause that they have less cognitive development than humans. Since PSD95p doesn't have a palmitoylation site, it cannot be located in the postsynaptic membrane for later anchoring of receptors and other proteins participating in the modulation of the synaptic force.
On the other hand, it is known that PSD95p can form supercomplexes with both NMDA and non-NMDA receptors 37 . These supercomplexes are the ARC protein (ARCp), which has been associated with learning processes and intelligence development. According to Fernandez et al. (2017), PSD95p is one of the proteins that interacts with ARCp in a superior percentage, finding that mutations in the PSD95p-ARCp complex can lead to a learning impairment dependent on the hippocampus 33 . The exciting results obtained in the present work could give the first evidence of improved intelligence development in humans compared to Neanderthals by allowing a modulation between LTP and LTD. This is necessary to decrease the synaptic connections between neurons that do not follow a sequential pattern due to the same stimulus, at the same time, increasing the strength of synaptic connections between neurons that are correlated 56 .
Finally, it has been established that the postsynaptic localization of AMPA receptors mediated by PSD95p plays a vital role in the regulation intracellular Ca + ion concentrations in postsynaptic cells 57,58 . The increase in Ca + is thought to be a generator of retrograde signaling that induces a more significant release of neurotransmitters from the presynaptic neuron to the synaptic space, leading to the same time to excitotoxicity 59 . Also, according to Zhang et al., (2014), the high concentrations of intracellular Ca + lead to the activation of the Ca + dependent calmodulin protein, which, when bound to the N-terminal end of PSD95p, inhibits the palmitoylation of PSD95p and, consequently, its dislocation from the postsynaptic membrane 60 .
As a result, higher production of NO is induced, subsequently resulting in the membrane's lipoperoxidation and destabilization of the postsynaptic receptors 59,61 . Consequently, pathologies, such as depression, post-traumatic stress disorder, and stroke are generated 62 . Therefore, it is suggested that proteolysis by the proteasome of human PSD95p induced by polyubiquitination could act as a regulatory mechanism in the disengagement of AMPA receptors to decrease the flow of Ca +2 ions dependent on NMDA receptors ( Figure. 6), favoring in this way the modulation of the synaptic force between neighboring neurons and so the increase of cognitive capacity.

Conclusions
In conclusion, these findings support the idea that the cognitive evolution of modern humans after the separation of our last common ancestor with Neanderthals and other hominid primates could be have been accompanied by discrete changes in the domain of polyubiquitination of PSD95p, which has a crucial role in the modulation of synaptic plasticity and cognitive development. However, it should be considered that PSD95p is not the only protein involved in synaptic plasticity processes. Therefore, to verify our claims, it is necessary to perform in vitro and in vivo experiments to evaluate that  The evolutionary history concerning the proteins was inferred by using the Maximum Likelihood method and Le_Gascuel_2008 model. The bootstrap consensus tree inferred from 2500 replicates both for genes and proteins represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. Percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (2500 replicates) are shown next to the branches.   .Regulation of synaptic strength by polyubiquitination of PSD95p. The polyubiquitination at the N-terminal end of PSD5p and subsequent proteolysis by the proteasome induces the delocalization and endocytosis of the AMPA receptors. This mechanism will decrease intracellularCa+ concentrations, modulating the synaptic connection between neurons that follow different activity profiles.