Human-specific features of spatial gene expression and regulation in eight brain regions.

Molecular maps of the human brain alone do not inform us of the features unique to humans. Yet, the identification of these features is important for understanding both the evolution and nature of human cognition. Here, we approached this question by analyzing gene expression and H3K27ac chromatin modification data collected in eight brain regions of humans, chimpanzees, gorillas, a gibbon, and macaques. An analysis of spatial transcriptome trajectories across eight brain regions in four primate species revealed 1851 genes showing human-specific transcriptome differences in one or multiple brain regions, in contrast to 240 chimpanzee-specific differences. More than half of these human-specific differences represented elevated expression of genes enriched in neuronal and astrocytic markers in the human hippocampus, whereas the rest were enriched in microglial markers and displayed human-specific expression in several frontal cortical regions and the cerebellum. An analysis of the predicted regulatory interactions driving these differences revealed the role of transcription factors in species-specific transcriptome changes, and epigenetic modifications were linked to spatial expression differences conserved across species.

Evolutionary acquisition of promoter-associated non-coding RNA (pancRNA) repertoires diversifies species-dependent gene activation mechanisms in mammals.

BACKGROUND: Recent transcriptome analyses have shown that long non-coding RNAs (ncRNAs) play extensive roles in transcriptional regulation. In particular, we have reported that promoter-associated ncRNAs (pancRNAs) activate the partner gene expression via local epigenetic changes. RESULTS: Here, we identify thousands of genes under pancRNA-mediated transcriptional activation in five mammalian species in common. In the mouse, 1) pancRNA-partnered genes confined their expression pattern to certain tissues compared to pancRNA-lacking genes, 2) expression of pancRNAs was significantly correlated with the enrichment of active chromatin marks, H3K4 trimethylation and H3K27 acetylation, at the promoter regions of the partner genes, 3) H3K4me1 marked the pancRNA-partnered genes regardless of their expression level, and 4) C- or G-skewed motifs were exclusively overrepresented between-200 and-1 bp relative to the transcription start sites of the pancRNA-partnered genes. More importantly, the comparative transcriptome analysis among five different mammalian species using a total of 25 counterpart tissues showed that the overall pancRNA expression profile exhibited extremely high species-specificity compared to that of total mRNA, suggesting that interspecies difference in pancRNA repertoires might lead to the diversification of mRNA expression profiles. CONCLUSIONS: The present study raises the interesting possibility that the gain and/or loss of gene-activation-associated pancRNA repertoires, caused by formation or disruption of the genomic GC-skewed structure in the course of evolution, finely shape the tissue-specific pattern of gene expression according to a given species.

Temporal plasticity involved in recovery from manual dexterity deficit after motor cortex lesion in macaque monkeys.

The question of how intensive motor training restores motor function after brain damage or stroke remains unresolved. Here we show that the ipsilesional ventral premotor cortex (PMv) and perilesional primary motor cortex (M1) of rhesus macaque monkeys are involved in the recovery of manual dexterity after a lesion of M1. A focal lesion of the hand digit area in M1 was made by means of ibotenic acid injection. This lesion initially caused flaccid paralysis in the contralateral hand but was followed by functional recovery of hand movements, including precision grip, during the course of daily postlesion motor training. Brain imaging of regional cerebral blood flow by means of H2 (15)O-positron emission tomography revealed enhanced activity of the PMv during the early postrecovery period and increased functional connectivity within M1 during the late postrecovery period. The causal role of these areas in motor recovery was confirmed by means of pharmacological inactivation by muscimol during the different recovery periods. These findings indicate that, in both the remaining primary motor and premotor cortical areas, time-dependent plastic changes in neural activity and connectivity are involved in functional recovery from the motor deficit caused by the M1 lesion. Therefore, it is likely that the PMv, an area distant from the core of the lesion, plays an important role during the early postrecovery period, whereas the perilesional M1 contributes to functional recovery especially during the late postrecovery period.

Deciphering social traits and pathophysiological conditions from natural behaviors in common marmosets.

Nonhuman primates (NHPs) are indispensable animal models by virtue of the continuity of behavioral repertoires across primates, including humans. However, behavioral assessment at the laboratory level has so far been limited. Employing the application of three-dimensional (3D) pose estimation and the optimal integration of subsequent analytic methodologies, we demonstrate that our artificial intelligence (AI)-based approach has successfully deciphered the ethological, cognitive, and pathological traits of common marmosets from their natural behaviors. By applying multiple deep neural networks trained with large-scale datasets, we established an evaluation system that could reconstruct and estimate the 3D poses of the marmosets, a small NHP that is suitable for analyzing complex natural behaviors in laboratory setups. We further developed downstream analytic methodologies to quantify a variety of behavioral parameters beyond motion kinematics. We revealed the distinct parental roles of male and female marmosets through automated detections of food-sharing behaviors using a spatial-temporal filter on 3D poses. Employing a recurrent neural network to analyze 3D pose time series data during social interactions, we additionally discovered that marmosets adjusted their behaviors based on others' internal state, which is not directly observable but can be inferred from the sequence of others' actions. Moreover, a fully unsupervised approach enabled us to detect progressively appearing symptomatic behaviors over a year in a Parkinson's disease model. The high-throughput and versatile nature of an AI-driven approach to analyze natural behaviors will open a new avenue for neuroscience research dealing with big-data analyses of social and pathophysiological behaviors in NHPs.

Effects of early versus late rehabilitative training on manual dexterity after corticospinal tract lesion in macaque monkeys.

Dexterous hand movements can be restored with motor rehabilitative training after a lesion of the lateral corticospinal tract (l-CST) in macaque monkeys. To maximize effectiveness, the optimal time to commence such rehabilitative training must be determined. We conducted behavioral analyses and compared the recovery of dexterous hand movements between monkeys in which hand motor training was initiated immediately after the l-CST lesion (early-trained monkeys) and those in which training was initiated 1 mo after the lesion (late-trained monkeys). The performance of dexterous hand movements was evaluated by food retrieval tasks. In early-trained monkeys, performance evaluated by the success rate in a vertical slit task (retrieval of a small piece of food through a narrow vertical slit) recovered to the level of intact monkeys during the first 1-2 mo after the lesion. In late-trained monkeys, the task success rate averaged ∼30% even after 3 mo of rehabilitative training. We also evaluated hand performance with the Klüver board task, in which monkeys retrieved small spherical food pellets from cylindrical wells. Although the success rate of the Klüver board task did not differ between early- and late-trained monkeys, kinematic movement analysis showed that there was a difference between the groups: late-trained monkeys with an improved success rate frequently used alternate movement strategies that were different from those used before the lesion. These results suggest that early rehabilitative training after a spinal cord lesion positively influences subsequent functional recovery.

Functional annotation of genes differentially expressed between primary motor and prefrontal association cortices of macaque brain.

DNA microarray-based genome-wide transcriptional profiling and gene network analyses were used to characterize the molecular underpinnings of the neocortical organization in rhesus macaque, with particular focus on the differences in the functional annotation of genes in the primary motor cortex (M1) and the prefrontal association cortex (area 46 of Brodmann). Functional annotation of the differentially expressed genes showed that the list of genes selectively expressed in M1 was enriched with genes involved in oligodendrocyte function, and energy consumption. The annotation appears to have successfully extracted the characteristics of the molecular structure of M1.

Diurnal variation in declarative memory and the involvement of SCOP in cognitive functions in nonhuman primates.

Cognitive functions depend on the time of day in various organisms. Previously, we found that 24-h recognition memory performance of nocturnal mice changes diurnally through SCOP protein-dependent regulation. It remains unknown whether diurnal change and SCOP-dependent regulation of memory performance are conserved across species with diurnal/nocturnal habits. We tested whether the memory performance of diurnal Japanese macaques depends on the time of day. The memory association between bitter taste of drinking water and the nozzle color of the water bottle was established during the light period of the day to evaluate of memory performance for macaques. Here we found diurnal variation of declarative memory in Japanese macaques. The middle of the daytime is the most effective time for memory performance during the light period. To assess whether SCOP is involved in declarative memory performance, we interfered with SCOP expression by using lentiviral vector expressing shRNA against Scop in the hippocampus of Japanese macaques. Scop knockdown in the hippocampus abrogated the memory performance in the middle of the daytime. Our results implicate that SCOP in the hippocampus is necessary for the diurnal rhythm of the memory system and that the SCOP-dependent memory regulation system may be conserved in mammals.

Reorganization of Corticospinal Projections after Prominent Recovery of Finger Dexterity from Partial Spinal Cord Injury in Macaque Monkeys.

We investigated morphologic changes in the corticospinal tract (CST) to understand the mechanism underlying recovery of hand function after lesion of the CST at the C4/C5 border in seven macaque monkeys. All monkeys exhibited prominent recovery of precision grip success ratio within a few months. The trajectories and terminals of CST from the contralesional (n = 4) and ipsilesional (n = 3) hand area of primary motor cortex (M1) were investigated at 5-29 months after the injury using an anterograde neural tracer, biotinylated dextran amine (BDA). Reorganization of the CST was assessed by counting the number of BDA-labeled axons and bouton-like swellings in the gray and white matters. Rostral to the lesion (at C3), the number of axon collaterals of the descending axons from both contralesional and ipsilesional M1 entering the ipsilesional and contralesional gray matter, respectively, were increased. Caudal to the lesion (at C8), axons originating from the contralesional M1, descending in the preserved gray matter around the lesion, and terminating in ipsilesional Laminae VI/VII and IX were observed. In addition, axons and terminals from the ipsilesional M1 increased in the ipsilesional Lamina IX after recrossing the midline, which were not observed in intact monkeys. Conversely, axons originating from the ipsilesional M1 and directed toward the contralesional Lamina VII decreased. These results suggest that multiple reorganizations of the corticospinal projections to spinal segments both rostral and caudal to the lesion originating from bilateral M1 underlie a prominent recovery in long-term after spinal cord injury.

Emergence of an erythroid cell-specific regulatory region in ABO intron 1 attributable to A- or B-antigen expression on erythrocytes in Hominoidea.

A- and B-antigens are present on red blood cells (RBCs) as well as other cells and secretions in Hominoidea including humans and apes such as chimpanzees and gibbons, whereas expression of these antigens on RBCs is subtle in monkeys such as Japanese macaques. Previous studies have indicated that H-antigen expression has not completely developed on RBCs in monkeys. Such antigen expression requires the presence of H-antigen and A- or B-transferase expression in cells of erythroid lineage, although whether or not ABO gene regulation is associated with the difference of A- or B-antigen expression between Hominoidea and monkeys has not been examined. Since it has been suggested that ABO expression on human erythrocytes is dependent upon an erythroid cell-specific regulatory region or the + 5.8-kb site in intron 1, we compared the sequences of ABO intron 1 among non-human primates, and demonstrated the presence of sites orthologous to the + 5.8-kb site in chimpanzees and gibbons, and their absence in Japanese macaques. In addition, luciferase assays revealed that the former orthologues enhanced promoter activity, whereas the corresponding site in the latter did not. These results suggested that the A- or B-antigens on RBCs might be ascribed to emergence of the + 5.8-kb site or the corresponding regions in ABO through genetic evolution.

Origin of Multisynaptic Corticospinal Pathway to Forelimb Segments in Macaques and Its Reorganization After Spinal Cord Injury.

Removal of the monosynaptic corticospinal pathway (CSP) terminating within the forelimb segments severely impairs manual dexterity. Functional recovery from the monosynaptic CSP lesion can be achieved through the remaining multisynaptic CSP toward the forelimb segments. In the present study, we applied retrograde transsynaptic labeling with rabies virus to a monkey model of spinal cord injury. By injecting the virus into the spinal forelimb segments immediately after the monosynaptic CSP lesion, we showed that the contralateral primary motor cortex (M1), especially its caudal and bank region (so-called "new" M1), was the principal origin of the CSP linking the motor cortex to the spinal forelimb segments disynaptically (disynaptic CSP). This forms a striking contrast to the architecture of the monosynaptic CSP that involves extensively other motor-related areas, together with M1. Next, the rabies injections were made at the recovery period of 3 months after the monosynaptic CSP lesion. The second-order labeled neurons were located in the ipsilateral as well as in the contralateral "new" M1. This indicates that the disynaptic CSP input from the ipsilateral "new" M1 is recruited during the motor recovery from the monosynaptic CSP lesion. Our results suggest that the disynaptic CSP is reorganized to connect the ipsilateral "new" M1 to the forelimb motoneurons for functional compensation after the monosynaptic CSP lesion.

[Species Differences in the Brain between Human and Non-Human Primates].

Cognitive abilities of humans are superior to that of non-human primates (NHP). Differences in genes, gene expression, cellular composition and response, neural circuit, and brain volume between humans and NHP underlying cognitive abilities are discussed, with a focus on cortical expansion, prefrontal expansion, language-related areas, and the adaptation of longer life spans in humans.

Neuronal and microglial localization of secreted phosphoprotein 1 (osteopontin) in intact and damaged motor cortex of macaques.

We previously reported that mRNA encoding secreted phosphoprotein 1 (SPP1), also known as osteopontin, is preferentially expressed in large neurons in layer V of the macaque motor cortex, most of which are presumed to be corticospinal tract neurons. As a first step to elucidating the cellular function of SPP1 in macaque neurons, we examined the localization of SPP1 in the primary motor cortex (M1) of the macaque by using immunohistochemistry. SPP1 immunoreactivity was found to be localized in the cell bodies of neurons, but not outside the cells, indicating that SPP1 was not secreted from these neurons. The results of electron microscope analysis and double-labeling analysis with marker proteins suggested that SPP1 was localized in the mitochondria of neurons. The distributions of SPP1 in the neurons corresponded to those of integrin αV, a putative receptor for SPP1. The distribution of SPP1 was also investigated in macaques whose M1 had been lesioned. We found that SPP1 was secreted by proliferated microglia in the lesioned area. Double-labeling analysis indicated that SPP1 immunoreactivity in the microglia was colocalized with CD44, another putative receptor for SPP1. Success rates in the small-object-retrieval task were positively correlated with SPP1 immunoreactivity in the neurons in the perilesional area. SPP1 has multiple roles in the macaque motor cortex, and it may be a key protein during recovery of hand movement after brain damage.

Development of the supralaryngeal vocal tract in Japanese macaques: implications for the evolution of the descent of the larynx.

The configuration of the supralaryngeal vocal tract depends on the nonuniform growth of the oral and pharyngeal portion. The human pharynx develops to form a unique configuration, with the epiglottis losing contact with the velum. This configuration develops from the great descent of the larynx relative to the palate, which is accomplished through both the descent of the laryngeal skeleton relative to the hyoid and the descent of the hyoid relative to the palate. Chimpanzees show both processes of laryngeal descent, as in humans, but the evolutionary path before the divergence of the human and chimpanzee lineages is unclear. The development of laryngeal descent in six living Japanese macaque monkeys, Macaca fuscata, was examined monthly during the first three years of life using magnetic resonance imaging, to delineate the present or absence of these two processes and their contributions to the development of the pharyngeal topology. The macaque shows descent of the hyoid relative to the palate, but lacks the descent of the laryngeal skeleton relative to the hyoid and that of the EG from the VL. We argue that the former descent is simply a morphological consequence of mandibular growth and that the latter pair of descents arose in a common ancestor of extant hominoids. Thus, the evolutionary path of the great descent of the larynx is likely to be explained by a model comprising multiple and mosaic evolutionary pathways, wherein these developmental phenomena may have contributed secondarily to the faculty of speech in the human lineage.

Age-related changes of elements in the coronary arteries of monkeys in comparison with those of humans.

To elucidate compositional changes of the coronary artery with aging, the authors investigated age-related changes of elements in the coronary arteries of rhesus and Japanese monkeys by direct chemical analysis in comparison with the coronary arteries of Japanese and Thai. Used monkeys consisted of 38 rhesus monkeys and 23 Japanese monkeys, ranging in age from newborn to 33 years. After perfusion with a fixative, the hearts were resected from the monkeys, and the anterior interventricular branches of the left coronary artery and the right coronary arteries were resected from the hearts. After ashing of the arteries, element contents were determined by inductively coupled plasma-atomic emission spectrometry. It was found that the Ca and P contents did not increase in both the left and right coronary arteries of rhesus and Japanese monkeys at old age. The average contents of Ca and P decreased by 13% and 25% in the left coronary arteries more than 20 years of age in comparison with those below 20 years of age, whereas they decreased by 4% and 15% in the right coronary arteries more than 20 years of age in comparison with those below 20 years of age. This finding indicated that atherosclerosis scarcely occurred in both the left and right coronary arteries of rhesus and Japanese monkeys at old age. In contrast with monkeys, atherosclerosis occurred frequently in the coronary arteries of Japanese and Thai at old age.

Comprehensive analysis of area-specific and time-dependent changes in gene expression in the motor cortex of macaque monkeys during recovery from spinal cord injury.

The present study aimed to assess the molecular bases of cortical compensatory mechanisms following spinal cord injury in primates. To accomplish this, comprehensive changes in gene expression were investigated in the bilateral primary motor cortex (M1), dorsal premotor cortex (PMd), and ventral premotor cortex (PMv) after a unilateral lesion of the lateral corticospinal tract (l-CST). At 2 weeks after the lesion, a large number of genes exhibited altered expression levels in the contralesional M1, which is directly linked to the lesioned l-CST. Gene ontology and network analyses indicated that these changes in gene expression are involved in the atrophy and plasticity changes observed in neurons. Orchestrated gene expression changes were present when behavioral recovery was attained 3 months after the lesion, particularly among the bilateral premotor areas, and a large number of these genes are involved in plasticity. Moreover, several genes abundantly expressed in M1 of intact monkeys were upregulated in both the PMd and PMv after the l-CST lesion. These area-specific and time-dependent changes in gene expression may underlie the molecular mechanisms of functional recovery following a lesion of the l-CST.

Expression of protein kinase-C substrate mRNA in the motor cortex of adult and infant macaque monkeys.

To understand the molecular and cellular bases of plasticity in the primate motor cortex, we investigated the expression of three protein kinase-C (PKC) substrates: GAP-43, myristoylated alanine-rich C-kinase substrate (MARCKS), and neurogranin, which are key molecules regulating synaptic plasticity. Prominent signals for the three mRNAs were primarily observed in pyramidal cells. Large pyramidal cells in layer V, from which the descending motor tract originates, contained weaker hybridization signals for GAP-43 and neurogranin mRNAs than did the smaller pyramidal cells. We also performed double-label in situ hybridization showing that GAP-43 and neurogranin mRNAs were expressed in a subset of MARCKS-positive neurons. Quantitative analysis showed that the expression was different between the layers: layer VI contained the strongest and layer II the weakest signals for all three mRNAs. The expression levels of GAP-43 and MARCKS mRNA in layer V were higher than in layer III, while the expression level of neurogranin mRNA in layer V was almost the same as in layer III. Developmental analysis from the newborn to adult indicated that the expression levels of the three mRNAs were higher in the infant motor cortex than in the adult. The expression of both GAP-43 and neurogranin mRNAs transiently increased over several months postnatally. The present study showed that the expression of the three PKC substrates was specific to cell types, cortical layers, and postnatal developmental stage. The specific expression may reflect functional specialization for plasticity in the motor cortex of both infants and adults.

Co-Opted Megasatellite DNA Drives Evolution of Secondary Night Vision in Azara's Owl Monkey.

Owl monkeys (genus Aotus) are the only taxon in simian primates that consists of nocturnal or otherwise cathemeral species. Their night vision is superior to that of other monkeys, apes, and humans but not as good as that of typical nocturnal mammals. This incomplete night vision has been used to conclude that these monkeys only secondarily adapted to a nocturnal lifestyle, or to their cathemeral lifestyle that involves high night-time activity. It is known that the rod cells of many nocturnal mammals possess a unique nuclear architecture in which heterochromatin is centrally located. This "inverted nuclear architecture", in contrast with "conventional nuclear architecture", provides elevated night vision by passing light efficiently to the outer segments of photoreceptors. Owl monkey rod cells exhibit an intermediate chromatin distribution, which may provide them with less efficient night vision than other nocturnal mammals. Recently, we identified three megasatellite DNAs in the genome of Azara's owl monkey (Aotus azarae). In the present study, we show that one of the three megasatellite DNAs, OwlRep, serves as the primary component of the heterochromatin block located in the central space of the rod nucleus in A. azarae. This satellite DNA is likely to have emerged in the Aotus lineage after its divergence from those of other platyrrhini taxa and underwent a rapid expansion in the genome. Our results indicate that the heterochromatin core in the A. azarae rod nucleus was newly formed in A. azarae or its recent ancestor, and supports the hypothesis that A. azarae, and with all probability other Aotus species, secondarily acquired night vision.

Expression of protein kinase C-substrate mRNAs in the basal ganglia of adult and infant macaque monkeys.

We performed in situ hybridization histochemistry on the monkey basal ganglia to investigate the mRNA localization of three protein kinase C substrates (GAP-43, MARCKS, and neurogranin), of which expression plays a role in structural changes in neurites and synapses. Weak hybridization signals for GAP-43 mRNA and intense signals for both MARCKS and neurogranin mRNAs were observed in the adult neostriatum. All three of the mRNAs were expressed in both substance P-positive direct pathway neurons and enkephalin-positive indirect pathway neurons. In the nucleus accumbens, the hybridization signals for the three mRNAs were weaker than those in the neostriatum. Double-label in situ hybridization histochemistry in the neostriatum revealed that GAP-43 and neurogranin mRNAs were expressed in a subset of MARCKS-positive neurons. While intense hybridization signals for MARCKS mRNA were observed in all of the other basal ganglia regions such as the globus pallidus, substantia innominata, subthalamic nucleus, and substantia nigra, intense signals for GAP-43 mRNA were restricted to the substantia innominata and substantia nigra pars compacta. No signal for neurogranin mRNA was observed in the basal ganglia regions outside the neostriatum and the nucleus accumbens. These results indicate that the protein kinase C substrates are abundant in some specific connections in cortico-basal ganglia circuits. Developmental analysis showed that the expression level in the putamen and nucleus accumbens, but not in the caudate nucleus, was higher in the infant than in the adult, suggesting that synaptic maturation in the caudate nucleus occurs earlier than that in the putamen and nucleus accumbens.

Northern blot and in situ hybridization analyses for the neurogranin mRNA in the developing monkey cerebral cortex.

Neurogranin is a postsynaptic substrate for protein kinase C, and its expression is related to dendritic spine development and postsynaptic plasticity. Using both Northern blot analysis and in situ hybridization techniques, we investigated the developmental changes of neurogranin expression in the monkey cerebral cortex. In each of four neocortical areas examined, i.e., the prefrontal area (area FD of von Bonin and Bailey), the temporal association area (TE), the primary somatosensory area (PB), and the primary visual area (OC), the Northern blot analysis showed that the amount of neurogranin mRNA was low during the prenatal and perinatal periods until postnatal day 8. It increased during postnatal development and reached its peak value at postnatal day 70 (in area OC) or postnatal month 6 (in area FD, TE, and PB). After that, the amount of neurogranin mRNA in the cerebral neocortex decreased gradually until postnatal years 2-3. The in situ hybridization experiments also showed a transient increase of neurogranin mRNA in the neocortex during postnatal day 70 to postnatal month 6. The transient increase was prominent in layers II and III of areas FD and TE; deep in layer III of area PB; and in layers II, III, and IV of area OC. In the hippocampus, in contrast to the results in the neocortex, the expression of neurogranin mRNA was decreased almost continuously during the postnatal period. The transiently increased expression of neurogranin in the postnatal neocortex may be a molecular basis for the postsynaptic modification of afferent inputs possibly from subcortical structures.

Age-dependent decreases of calcium, phosphorus, sulfur, and zinc in the cardiac valves of monkeys.

To elucidate compositional changes of the cardiac valves in monkey with aging, the authors investigated age-related changes of elements in the four cardiac valves of rhesus and Japanese monkeys and the relationships among elements by inductively coupled plasma-atomic emission spectrometry (ICP-AES). The four cardiac valves of the aortic, pulmonary, mitral, and tricuspid valves were resected from 19 rhesus and 11 Japanese monkeys, ranging in age from 10 d to 33 yr. The element contents were determined by ICP-AES. It was found that the Ca, P, S, and Zn contents were high in the four cardiac valves of the monkeys below 1 yr and thereafter decreased significantly with aging, except for Ca in the mitral valve, for which no significant correlation was found between age and Ca content. The Ca and P contents did not increase in the four cardiac valves at old age. This result revealed that calcification scarcely occurred in the four cardiac valves at old age. With regard to the relationships among elements, it was found that there were significant direct correlations among the Ca, P, S, and Zn contents in all of the four cardiac valves of the monkeys, with two exceptions between P and Zn contents in both the aortic and pulmonary valves. Therefore, as Ca decreased in the four cardiac valves, P, S, and Zn decreased simultaneously in the same cardiac valves.