Perinatal interference with the serotonergic system affects VTA function in the adult via glutamate co-transmission.

Serotonin and dopamine are associated with multiple psychiatric disorders. How they interact during development to affect subsequent behavior remains unknown. Knockout of the serotonin transporter or postnatal blockade with selective serotonin reuptake inhibitors (SSRIs) leads to novelty-induced exploration deficits in adulthood, potentially involving the dopamine system. Here, we show in the mouse that raphe nucleus serotonin neurons activate ventral tegmental area dopamine neurons via glutamate co-transmission and that this co-transmission is reduced in animals exposed postnatally to SSRIs. Blocking serotonin neuron glutamate co-transmission mimics this SSRI-induced hypolocomotion, while optogenetic activation of dopamine neurons reverses this hypolocomotor phenotype. Our data demonstrate that serotonin neurons modulate dopamine neuron activity via glutamate co-transmission and that this pathway is developmentally malleable, with high serotonin levels during early life reducing co-transmission, revealing the basis for the reduced novelty-induced exploration in adulthood due to postnatal SSRI exposure.

Increased neuronal expression of the early endosomal adaptor APPL1 leads to endosomal and synaptic dysfunction with cholinergic neurodegeneration.

Dysfunction of the endolysosomal system within neurons is a prominent feature of Alzheimer's disease (AD) pathology. Multiple AD-risk factors are known to cause hyper-activity of the early-endosome small GTPase rab5, resulting in neuronal endosomal pathway disruption. APPL1, an important rab5 effector protein, is an interface between endosomal and neuronal function through a rab5-activating interaction with the BACE1-generated C-terminal fragment (ßCTF or C99) of the amyloid precursor protein (APP), a pathogenic APP fragment generated within endolysosomal compartments. To better understand the role of APPL1 in the AD endosomal phenotype, we generated a transgenic mouse model over-expressing human APPL1 within neurons (Thy1-APPL1 mice). Consistent with the important endosomal regulatory role of APPL1, Thy1-APPL1 mice have enlarged neuronal early endosomes and increased synaptic endocytosis due to increased rab5 activation. We additionally demonstrate pathological consequences of APPL1 overexpression, including functional changes in hippocampal long-term potentiation (LTP) and long-term depression (LTD), as well as degeneration of the large projection cholinergic neurons of the basal forebrain and impairment of hippocampal-dependent memory. Our findings show that increased neuronal APPL1 levels lead to a cascade of pathological effects within neurons, including early endosomal alterations, synaptic dysfunction, and neurodegeneration. Multiple risk factors and molecular regulators, including APPL1 activity, are known to contribute to the endosomal dysregulation seen in the early stages of AD, and these findings further highlight the shared pathobiology and consequences to a neuron of early endosomal pathway disruption. Significance Statement: Dysfunction in the endolysosomal system within neurons is a key feature of Alzheimer's disease (AD). Multiple AD risk factors lead to hyperactivity of the early-endosome GTPase rab5, disrupting neuronal pathways including the cholinergic circuits involved early in memory decline. APPL1, a crucial rab5 effector, connects endosomal and neuronal functions through its interaction with a specific amyloid precursor protein (APP) fragment generated within endosomes. To understand APPL1's role, a transgenic mouse model over-expressing human APPL1 in neurons (Thy1-APPL1 mice) was developed. These mice show enlarged early endosomes and increased synaptic endocytosis due to rab5 activation, resulting in impaired hippocampal long-term potentiation and depression, the degeneration of basal forebrain cholinergic neurons, and memory deficits, highlighting a pathological cascade mediated through APPL1 at the early endosome.

Estimates of total neuron number show that neonatal ethanol causes immediate and lasting neuron loss in cortical and subcortical areas.

In neonatal brain development there is a period of normal apoptotic cell death that regulates adult neuron number. At approximately the same period, ethanol exposure can cause a dramatic spike in apoptotic cell death. While ethanol-induced apoptosis has been shown to reduce adult neuron number, questions remain about the regional selectivity of the ethanol effect, and whether the brain might have some capacity to overcome the initial neuron loss. The present study used stereological cell counting to compare cumulative neuron loss 8 h after postnatal day 7 (P7) ethanol treatment to that of animals left to mature to adulthood (P70). Across several brain regions we found that the reduction of total neuron number after 8 h was as large as that of adult animals. Comparison between regions revealed that some areas are more vulnerable, with neuron loss in the anterior thalamic nuclei > the medial septum/vertical diagonal band, dorsal subiculum, and dorsal lateral geniculate nucleus > the mammillary bodies and cingulate cortex > whole neocortex. In contrast to estimates of total neuron number, estimates of apoptotic cell number in Nissl-stained sections at 8 h after ethanol treatment provided a less reliable predictor of adult neuron loss. The findings show that ethanol-induced neonatal apoptosis often causes immediate neuron deficits that persist in adulthood, and furthermore suggests that the brain may have limited capacity to compensate for ethanol-induced neuron loss.

Faulty autolysosome acidification in Alzheimer's disease mouse models induces autophagic build-up of Aß in neurons, yielding senile plaques.

Autophagy is markedly impaired in Alzheimer's disease (AD). Here we reveal unique autophagy dysregulation within neurons in five AD mouse models in vivo and identify its basis using a neuron-specific transgenic mRFP-eGFP-LC3 probe of autophagy and pH, multiplex confocal imaging and correlative light electron microscopy. Autolysosome acidification declines in neurons well before extracellular amyloid deposition, associated with markedly lowered vATPase activity and build-up of Aß/APP-ßCTF selectively within enlarged de-acidified autolysosomes. In more compromised yet still intact neurons, profuse Aß-positive autophagic vacuoles (AVs) pack into large membrane blebs forming flower-like perikaryal rosettes. This unique pattern, termed PANTHOS (poisonous anthos (flower)), is also present in AD brains. Additional AVs coalesce into peri-nuclear networks of membrane tubules where fibrillar ß-amyloid accumulates intraluminally. Lysosomal membrane permeabilization, cathepsin release and lysosomal cell death ensue, accompanied by microglial invasion. Quantitative analyses confirm that individual neurons exhibiting PANTHOS are the principal source of senile plaques in amyloid precursor protein AD models.

Preclinical and randomized clinical evaluation of the p38α kinase inhibitor neflamapimod for basal forebrain cholinergic degeneration.

The endosome-associated GTPase Rab5 is a central player in the molecular mechanisms leading to degeneration of basal forebrain cholinergic neurons (BFCN), a long-standing target for drug development. As p38α is a Rab5 activator, we hypothesized that inhibition of this kinase holds potential as an approach to treat diseases associated with BFCN loss. Herein, we report that neflamapimod (oral small molecule p38α inhibitor) reduces Rab5 activity, reverses endosomal pathology, and restores the numbers and morphology of BFCNs in a mouse model that develops BFCN degeneration. We also report on the results of an exploratory (hypothesis-generating) phase 2a randomized double-blind 16-week placebo-controlled clinical trial (Clinical trial registration: NCT04001517/EudraCT #2019-001566-15) of neflamapimod in mild-to-moderate dementia with Lewy bodies (DLB), a disease in which BFCN degeneration is an important driver of disease expression. A total of 91 participants, all receiving background cholinesterase inhibitor therapy, were randomized 1:1 between neflamapimod 40 mg or matching placebo capsules (taken orally twice-daily if weight <80 kg or thrice-daily if weight >80 kg). Neflamapimod does not show an effect in the clinical study on the primary endpoint, a cognitive-test battery. On two secondary endpoints, a measure of functional mobility and a dementia rating-scale, improvements were seen that are consistent with an effect on BFCN function. Neflamapimod treatment is well-tolerated with no study drug associated treatment discontinuations. The combined preclinical and clinical observations inform on the validity of the Rab5-based pathogenic model of cholinergic degeneration and provide a foundation for confirmatory (hypothesis-testing) clinical evaluation of neflamapimod in DLB.

Hemispheric comparisons of neuron density in the planum temporale of schizophrenia and nonpsychiatric brains.

Postmortem and in vivo studies of schizophrenia frequently reveal reduced cortical volume, but the underlying cellular abnormalities are incompletely defined. One influential hypothesis, especially investigated in Brodmann's area 9 of prefrontal cortex, is that the number of neurons is normal, and the volume change is caused by reduction of the surrounding neuropil. However, studies have differed on whether the cortex has the increased neuron density that is predicted by this hypothesis. In a recent study of bilateral planum temporale (PT), we reported smaller volume and width of the outer cortex (layers I-III), especially in the left hemisphere, among subjects with schizophrenia. In the present study, we measured neuron density and size in the same PT samples, and also in prefrontal area 9 of the same brains. In the PT, separate stereological measurements were made in layers II, IIIc, and VI, whereas area 9 was sampled in layer IIIb-c. In both cortical regions, there was no significant effect of schizophrenia on neuronal density or size. There was, nevertheless, a trend-level right>left hemispheric asymmetry of neuron density in the PT, which may partially explain the previously reported left>right asymmetry of cortical width. In schizophrenia, our findings suggest that closer packing of neurons may not always explain reduced cortical volume, and subtly decreased neuron number may be a contributing factor.

Neonatal ethanol causes profound reduction of cholinergic cell number in the basal forebrain of adult animals.

In animal models that mimic human third-trimester fetal development, ethanol causes substantial cellular apoptosis in the brain, but for most brain structures, the extent of permanent neuron loss that persists into adulthood is unknown. We injected ethanol into C57BL/6J mouse pups at postnatal day 7 (P7) to model human late-gestation ethanol toxicity, and then used stereological methods to investigate adult cell numbers in several subcortical neurotransmitter systems that project extensively in the forebrain to regulate arousal states. Ethanol treatment caused especially large reductions (34-42%) in the cholinergic cells of the basal forebrain, including cholinergic cells in the medial septal/vertical diagonal band nuclei (Ch1/Ch2) and in the horizontal diagonal band/substantia innominata/nucleus basalis nuclei (Ch3/Ch4). Cell loss was also present in non-cholinergic basal forebrain cells, as demonstrated by 34% reduction of parvalbumin-immunolabeled GABA cells and 25% reduction of total Nissl-stained neurons in the Ch1/Ch2 region. In contrast, cholinergic cells in the striatum were reduced only 12% by ethanol, and those of the brainstem pedunculopontine/lateral dorsal tegmental nuclei (Ch5/Ch6) were not significantly reduced. Similarly, ethanol did not significantly reduce dopamine cells of the ventral tegmental area/substantia nigra or serotonin cells in the dorsal raphe nucleus. Orexin (hypocretin) cells in the hypothalamus showed a modest reduction (14%). Our findings indicate that the basal forebrain is especially vulnerable to alcohol exposure in the late gestational period. Reduction of cholinergic and GABAergic projection neurons from the basal forebrain that regulate forebrain arousal may contribute to the behavioral and cognitive deficits associated with neonatal ethanol exposure.

Endosomal Dysfunction Induced by Directly Overactivating Rab5 Recapitulates Prodromal and Neurodegenerative Features of Alzheimer's Disease.

Neuronal endosomal dysfunction, the earliest known pathobiology specific to Alzheimer's disease (AD), is mediated by the aberrant activation of Rab5 triggered by APP-ß secretase cleaved C-terminal fragment (APP-ßCTF). To distinguish pathophysiological consequences specific to overactivated Rab5 itself, we activate Rab5 independently from APP-ßCTF in the PA-Rab5 mouse model. We report that Rab5 overactivation alone recapitulates diverse prodromal and degenerative features of AD. Modest neuron-specific transgenic Rab5 expression inducing hyperactivation of Rab5 comparable to that in AD brain reproduces AD-related Rab5-endosomal enlargement and mistrafficking, hippocampal synaptic plasticity deficits via accelerated AMPAR endocytosis and dendritic spine loss, and tau hyperphosphorylation via activated glycogen synthase kinase-3ß. Importantly, Rab5-mediated endosomal dysfunction induces progressive cholinergic neurodegeneration and impairs hippocampal-dependent memory. Aberrant neuronal Rab5-endosome signaling, therefore, drives a pathogenic cascade distinct from ß-amyloid-related neurotoxicity, which includes prodromal and neurodegenerative features of AD, and suggests Rab5 overactivation as a potential therapeutic target.

Effects of neonatal ethanol on cerebral cortex development through adolescence.

Neonatal brain lesions cause deficits in structure and function of the cerebral cortex that sometimes are not fully expressed until adolescence. To better understand the onset and persistence of changes caused by postnatal day 7 (P7) ethanol treatment, we examined neocortical cell numbers, volume, surface area and thickness from neonatal to post-adolescent ages. In control mice, total neuron number decreased from P8 to reach approximately stable levels at about P30, as expected from normal programmed cell death. Cortical thickness reached adult levels by P14, but cortical volume and surface area continued to increase from juvenile (P20-30) to post-adolescent (P54-93) ages. P7 ethanol caused a reduction of total neurons by P14, but this deficit was transient, with later ages having only small and non-significant reductions. Previous studies also reported transient neuron loss after neonatal lesions that might be partially explained by an acute acceleration of normally occurring programmed cell death. GABAergic neurons expressing parvalbumin, calretinin, or somatostatin were reduced by P14, but unlike total neurons the reductions persisted or increased in later ages. Cortical volume, surface area and thickness were also reduced by P7 ethanol. Cortical volume showed evidence of a transient reduction at P14, and then was reduced again in post-adolescent ages. The results show a developmental sequence of neonatal ethanol effects. By juvenile ages the cortex overcomes the P14 deficit of total neurons, whereas P14 GABA cell deficits persist. Cortical volume reductions were present at P14, and again in post-adolescent ages.

Neurofilament light interaction with GluN1 modulates neurotransmission and schizophrenia-associated behaviors.

Neurofilament (NFL) proteins have recently been found to play unique roles in synapses. NFL is known to interact with the GluN1 subunit of N-methyl-D-aspartic acid (NMDAR) and be reduced in schizophrenia though functional consequences are unknown. Here we investigated whether the interaction of NFL with GluN1 modulates synaptic transmission and schizophrenia-associated behaviors. The interaction of NFL with GluN1 was assessed by means of molecular, pharmacological, electrophysiological, magnetic resonance spectroscopy (MRS), and schizophrenia-associated behavior analyses. NFL deficits cause an NMDAR hypofunction phenotype including abnormal hippocampal function, as seen in schizophrenia. NFL-/- deletion in mice reduces dendritic spines and GluN1 protein levels, elevates ubiquitin-dependent turnover of GluN1 and hippocampal glutamate measured by MRS, and depresses hippocampal long-term potentiation. NMDAR-related behaviors are also impaired, including pup retrieval, spatial and social memory, prepulse inhibition, night-time activity, and response to NMDAR antagonist, whereas motor deficits are minimal. Importantly, partially lowering NFL in NFL+/- mice to levels seen regionally in schizophrenia, induced similar but milder NMDAR-related synaptic and behavioral deficits. Our findings support an emerging view that central nervous system neurofilament subunits including NFL in the present report, serve distinctive, critical roles in synapses relevant to neuropsychiatric diseases.

Reduced GABA neuron density in auditory cerebral cortex of subjects with major depressive disorder.

Although major depressive disorder (MDD) and schizophrenia (SZ) are closely associated with disrupted functions in frontal and limbic areas of cerebral cortex, cellular pathology has also been found in other brain areas, including primary sensory cortex. Auditory cortex is of particular interest, given the prominence of auditory hallucinations in SZ, and sensory deficits in MDD. We used stereological sampling methods in auditory cortex to look for cellular differences between MDD, SZ and non-psychiatric subjects. Additionally, as all of our MDD subjects died of suicide, we evaluated the association of suicide with our measurements by selecting a SZ sample that was divided between suicide and non-suicide subjects. Measurements were done in primary auditory cortex (area A1) and auditory association cortex (area Tpt), two areas with distinct roles in sensory processing and obvious differences in neuron density and size. In MDD, densities of GABAergic interneurons immunolabeled for calretinin (CR) and calbindin (CB) were 23-29% lower than non-psychiatric controls in both areas. Parvalbumin (PV) interneurons (counted only in area Tpt) showed a nominally smaller (16%) reduction that was not statistically significant. Total neuron and glia densities measured in Nissl stained sections did not show corresponding reductions. Analysis of suicide in the SZ sample indicated that reduced CR cell density was associated with suicide, whereas the densities of CB and other cells were not. Our results are consistent with previous studies in MDD that found altered GABA-associated markers throughout the cerebral cortex including primary sensory areas.

Partial BACE1 reduction in a Down syndrome mouse model blocks Alzheimer-related endosomal anomalies and cholinergic neurodegeneration: role of APP-CTF.

ß-amyloid precursor protein (APP) and amyloid beta peptide (Aß) are strongly implicated in Alzheimer's disease (AD) pathogenesis, although recent evidence has linked APP-ßCTF generated by BACE1 (ß-APP cleaving enzyme 1) to the development of endocytic abnormalities and cholinergic neurodegeneration in early AD. We show that partial BACE1 genetic reduction prevents these AD-related pathological features in the Ts2 mouse model of Down syndrome. Partially reducing BACE1 by deleting one BACE1 allele blocked development of age-related endosome enlargement in the medial septal nucleus, cerebral cortex, and hippocampus and loss of choline acetyltransferase (ChAT)-positive medial septal nucleus neurons. BACE1 reduction normalized APP-ßCTF elevation but did not alter Aß40 and Aß42 peptide levels in brain, supporting a critical role in vivo for APP-ßCTF in the development of these abnormalities. Although ameliorative effects of BACE1 inhibition on ß-amyloidosis and synaptic proteins levels have been previously noted in AD mouse models, our results highlight the additional potential value of BACE1 modulation in therapeutic targeting of endocytic dysfunction and cholinergic neurodegeneration in Down syndrome and AD.

Selective reduction of cerebral cortex GABA neurons in a late gestation model of fetal alcohol spectrum disorder.

Fetal alcohol spectrum disorders (FASD) are associated with cognitive and behavioral deficits, and decreased volume of the whole brain and cerebral cortex. Rodent models have shown that early postnatal treatments, which mimic ethanol toxicity in the third trimester of human pregnancy, acutely induce widespread apoptotic neuronal degeneration and permanent behavioral deficits. However, the lasting cellular and anatomical effects of early ethanol treatments are still incompletely understood. This study examined changes in neocortex volume, thickness, and cellular organization that persist in adult mice after postnatal day 7 (P7) ethanol treatment. Post mortem brain volumes, measured by both MRI within the skull and by fluid displacement of isolated brains, were reduced 10-13% by ethanol treatment. The cerebral cortex showed a similar reduction (12%) caused mainly by lower surface area (9%). In spite of these large changes, several features of cortical organization showed little evidence of change, including cortical thickness, overall neuron size, and laminar organization. Estimates of total neuron number showed a trend level reduction of about 8%, due mainly to reduced cortical volume but unchanged neuron density. However, counts of calretinin (CR) and parvalbumin (PV) subtypes of GABAergic neurons showed a striking >30% reduction of neuron number. Similar ethanol effects were found in male and female mice, and in C57BL/6By and BALB/cJ mouse strains. Our findings indicate that the cortex has substantial capacity to develop normal cytoarchitectonic organization after early postnatal ethanol toxicity, but there is a selective and persistent reduction of GABA cells that may contribute to the lasting cognitive and behavioral deficits in FASD.

Hemispheric asymmetry of primary auditory cortex and Heschl's gyrus in schizophrenia and nonpsychiatric brains.

Heschl's gyrus (HG) is reported to have a normal left>right hemispheric volume asymmetry, and reduced asymmetry in schizophrenia. Primary auditory cortex (A1) occupies the caudal-medial surface of HG, but it is unclear if A1 has normal asymmetry, or whether its asymmetry is altered in schizophrenia. To address these issues, we compared bilateral gray matter volumes of HG and A1, and neuron density and number in A1, in autopsy brains from male subjects with or without schizophrenia. Comparison of diagnostic groups did not reveal altered gray matter volumes, neuron density, neuron number or hemispheric asymmetries in schizophrenia. With respect to hemispheric differences, HG displayed a clear left>right asymmetry of gray matter volume. Area A1 occupied nearly half of HG, but had less consistent volume asymmetry, that was clearly present only in a subgroup of archival brains from elderly subjects. Neuron counts, in layers IIIb-c and V-VI, showed that the A1 volume asymmetry reflected differences in neuron number, and was not caused simply by changes in neuron density. Our findings confirm previous reports of striking hemispheric asymmetry of HG, and additionally show evidence that A1 has a corresponding asymmetry, although less consistent than that of HG.

Embedding matrix for simultaneous processing of multiple histological samples.

A method is described for simultaneous histological processing of multiple fixed tissue samples. The tissue samples are embedded in a gelatin-albumin protein matrix that is rapidly solidified and bound to the tissue surface using a cross-linking reagent. After freezing and sectioning, the individual sections containing multiple samples can be processed for immunocytochemical and histochemical staining. The method is demonstrated for simultaneous processing of multiple rodent brains, and for reconstruction of fragmented human postmortem brain samples.

Cortical thickness, neuron density and size in the inferior parietal lobe in schizophrenia.

The inferior parietal lobe (IPL) is a center of multisensory integration, and both functional and structural MRI studies have found evidence that pathology in this region may contribute to disrupted sensory perception in schizophrenia. To further define this pathology, we used postmortem samples from the left and right IPL, to compare the thickness and volume of the upper (I-III) and lower (IV-VI) cortical layers. The samples were divided into supramarginal and angular gyri, and neuron density and size were measured in the supramarginal gyrus. The laminar thickness and volume measurements did not demonstrate significant changes in schizophrenia, but did show that the angular gyrus was thinner than the supramarginal gyrus, due to a difference mainly in the lower layers. Measurements of cortical thickness, neuron size and neuron density all showed some evidence of previously reported normal hemispheric differences. These asymmetries were reduced in schizophrenia, but the small changes were at the threshold of detection, and are discussed in the context of the sensitivity of the methods applied.