Engineered neurogenesis in naïve adult rat cortex by Ngn2-mediated neuronal reprogramming of resident oligodendrocyte progenitor cells.

Adult tissue stem cells contribute to tissue homeostasis and repair but the long-lived neurons in the human adult cerebral cortex are not replaced, despite evidence for a limited regenerative response. However, the adult cortex contains a population of proliferating oligodendrocyte progenitor cells (OPCs). We examined the capacity of rat cortical OPCs to be re-specified to a neuronal lineage both in vitro and in vivo. Expressing the developmental transcription factor Neurogenin2 (Ngn2) in OPCs isolated from adult rat cortex resulted in their expression of early neuronal lineage markers and genes while downregulating expression of OPC markers and genes. Ngn2 induced progression through a neuronal lineage to express mature neuronal markers and functional activity as glutamatergic neurons. In vivo retroviral gene delivery of Ngn2 to naive adult rat cortex ensured restricted targeting to proliferating OPCs. Ngn2 expression in OPCs resulted in their lineage re-specification and transition through an immature neuronal morphology into mature pyramidal cortical neurons with spiny dendrites, axons, synaptic contacts, and subtype specification matching local cytoarchitecture. Lineage re-specification of rat cortical OPCs occurred without prior injury, demonstrating these glial progenitor cells need not be put into a reactive state to achieve lineage reprogramming. These results show it may be feasible to precisely engineer additional neurons directly in adult cerebral cortex for experimental study or potentially for therapeutic use to modify dysfunctional or damaged circuitry.

Protein mishandling and impaired lysosomal proteolysis generated through calcium dysregulation in Alzheimer's disease.

Impairments in neural lysosomal- and autophagic-mediated degradation of cellular debris contribute to neuritic dystrophy and synaptic loss. While these are well-characterized features of neurodegenerative disorders such as Alzheimer's disease (AD), the upstream cellular processes driving deficits in pathogenic protein mishandling are less understood. Using a series of fluorescent biosensors and optical imaging in model cells, AD mouse models and human neurons derived from AD patients, we reveal a previously undescribed cellular signaling cascade underlying protein mishandling mediated by intracellular calcium dysregulation, an early component of AD pathogenesis. Increased Ca2+ release via the endoplasmic reticulum (ER)-resident ryanodine receptor (RyR) is associated with reduced expression of the lysosome proton pump vacuolar-ATPase (vATPase) subunits (V1B2 and V0a1), resulting in lysosome deacidification and disrupted proteolytic activity in AD mouse models and human-induced neurons (HiN). As a result of impaired lysosome digestive capacity, mature autophagosomes with hyperphosphorylated tau accumulated in AD murine neurons and AD HiN, exacerbating proteinopathy. Normalizing AD-associated aberrant RyR-Ca2+ signaling with the negative allosteric modulator, dantrolene (Ryanodex), restored vATPase levels, lysosomal acidification and proteolytic activity, and autophagic clearance of intracellular protein aggregates in AD neurons. These results highlight that prior to overt AD histopathology or cognitive deficits, aberrant upstream Ca2+ signaling disrupts lysosomal acidification and contributes to pathological accumulation of intracellular protein aggregates. Importantly, this is demonstrated in animal models of AD, and in human iPSC-derived neurons from AD patients. Furthermore, pharmacological suppression of RyR-Ca2+ release rescued proteolytic function, revealing a target for therapeutic intervention that has demonstrated effects in clinically-relevant assays.

Induced Neurons for Disease Modeling and Repair: A Focus on Non-fibroblastic Cell Sources in Direct Reprogramming.

Direct cellular reprogramming exhibits distinct advantages over reprogramming from an induced pluripotent stem cell intermediate. These include a reduced risk of tumorigenesis and the likely preservation of epigenetic data. In vitro direct reprogramming approaches primarily aim to model the pathophysiological development of neurological disease and identify therapeutic targets, while in vivo direct reprogramming aims to develop treatments for various neurological disorders, including cerebral injury and cancer. In both approaches, there is progress toward developing increased control of subtype-specific production of induced neurons. A majority of research primarily utilizes fibroblasts as the donor cells. However, there are a variety of other somatic cell types that have demonstrated the potential for reprogramming into induced neurons. This review highlights studies that utilize non-fibroblastic cell sources for reprogramming, such as astrocytes, olfactory ensheathing cells, peripheral blood cells, Müller glia, and more. We will examine benefits and obstructions for translation into therapeutics or disease modeling, as well as efficiency of the conversion. A summary of donor cells, induced neuron types, and methods of induction is also provided.

Assessment of the Effects of Altered Amyloid-Beta Clearance on Behavior following Repeat Closed-Head Brain Injury in Amyloid-Beta Precursor Protein Humanized Mice.

Traumatic brain injury (TBI) increases the risk for dementias including Alzheimer's disease (AD) and chronic traumatic encephalopathy. Further, both human and animal model data indicate that amyloid-beta (Aß) peptide accumulation and its production machinery are upregulated by TBI. Considering the clear link between chronic Aß elevation and AD as well as tau pathology, the role(s) of Aß in TBI is of high importance. Endopeptidases, including the neprilysin (NEP)-like enzymes, are key mediators of Aß clearance and may affect susceptibility to pathology post-TBI. Here, we use a "humanized" mouse model of Aß production, which expresses normal human amyloid-beta precursor protein (APP) under its natural transcriptional regulation and exposed them to a more clinically relevant repeated closed-head TBI paradigm. These transgenic mice also were crossed with mice deficient for the Aß degrading enzymes NEP or NEP2 to assess models of reduced cerebral Aß clearance in our TBI model. Our results show that the presence of the human form of Aß did not exacerbate motor (Rotarod) and spatial learning/memory deficits (Morris water maze) post-injuries, while potentially reduced anxiety (Open Field) was observed. NEP and NEP2 deficiency also did not exacerbate these deficits post-injuries and was associated with protection from motor (NEP and NEP2) and spatial learning/memory deficits (NEP only). These data suggest that normally regulated expression of wild-type human APP/Aß does not contribute to deficits acutely after TBI and may be protective at this stage of injury.

Human-Induced Neurons from Presenilin 1 Mutant Patients Model Aspects of Alzheimer's Disease Pathology.

Traditional approaches to studying Alzheimer's disease (AD) using mouse models and cell lines have advanced our understanding of AD pathogenesis. However, with the growing divide between model systems and clinical therapeutic outcomes, the limitations of these approaches are increasingly apparent. Thus, to generate more clinically relevant systems that capture pathological cascades within human neurons, we generated human-induced neurons (HiNs) from AD and non-AD individuals to model cell autonomous disease properties. We selected an AD patient population expressing mutations in presenilin 1 (mPS1), which is linked to increased amyloid production, tau pathology, and calcium signaling abnormalities, among other features. While these AD components are detailed in model systems, they have yet to be collectively identified in human neurons. Thus, we conducted molecular, immune-based, electrophysiological, and calcium imaging studies to establish patterns of cellular pathology in this patient population. We found that mPS1 HiNs generate increased Aß42 and hyperphosphorylated tau species relative to non-AD controls, and exaggerated ER calcium responses that are normalized with ryanodine receptor (RyR) negative allosteric modulators. The inflammasome product, interleukin-18 (IL-18), also increased PS1 expression. This work highlights the potential for HiNs to model AD pathology and validates their role in defining cellular pathogenesis and their utility for therapeutic screening.

Targeting Amyloid-ß Precursor Protein, APP, Splicing with Antisense Oligonucleotides Reduces Toxic Amyloid-ß Production.

Alterations in amyloid beta precursor protein (APP) have been implicated in cognitive decline in Alzheimer's disease (AD), which is accelerated in Down syndrome/Trisomy 21 (DS/TS21), likely due to the extra copy of the APP gene, located on chromosome 21. Proteolytic cleavage of APP generates amyloid-ß (Aß) peptide, the primary component of senile plaques associated with AD. Reducing Aß production is predicted to lower plaque burden and mitigate AD symptoms. Here, we designed a splice-switching antisense oligonucleotide (SSO) that causes skipping of the APP exon that encodes proteolytic cleavage sites required for Aß peptide production. The SSO induced exon skipping in Down syndrome cell lines, resulting in a reduction of Aß. Treatment of mice with the SSO resulted in widespread distribution in the brain accompanied by APP exon skipping and a reduction of Aß. Overall, we show that an alternatively spliced isoform of APP encodes a cleavage-incompetent protein that does not produce Aß peptide and that promoting the production of this isoform with an SSO can reduce Aß in vivo. These findings demonstrate the utility of using SSOs to induce a spliced isoform of APP to reduce Aß as a potential approach for treating AD.

NPY Induces Stress Resilience via Downregulation of Ih in Principal Neurons of Rat Basolateral Amygdala.

Neuropeptide Y (NPY) expression is tightly linked with the development of stress resilience in rodents and humans. Local NPY injections targeting the basolateral amygdala (BLA) produce long-term behavioral stress resilience in male rats via an unknown mechanism. Previously, we showed that activation of NPY Y1 receptors hyperpolarizes BLA principal neurons (PNs) through inhibition of the hyperpolarization-activated, depolarizing H-current, Ih The present studies tested whether NPY treatment induces stress resilience by modulating Ih NPY (10 pmol) was delivered daily for 5 d bilaterally into the BLA to induce resilience; thereafter, the electrophysiological properties of PNs and the expression of Ih in the BLA were characterized. As reported previously, increases in social interaction (SI) times persisted weeks after completion of NPY administration. In vitro intracellular recordings showed that repeated intra-BLA NPY injections resulted in hyperpolarization of BLA PNs at 2 weeks (2W) and 4 weeks (4W) after NPY treatment. At 2W, spontaneous IPSC frequencies were increased, whereas at 4W, resting Ih was markedly reduced and accompanied by decreased levels of HCN1 mRNA and protein expression in BLA. Knock-down of HCN1 channels in the BLA with targeted delivery of lentivirus containing HCN1-shRNA increased SI beginning 2W after injection and induced stress resilience. NPY treatment induced sequential, complementary changes in the inputs to BLA PNs and their postsynaptic properties that reduce excitability, a mechanism that contributes to less anxious behavior. Furthermore, HCN1 knock-down mimicked the increases in SI and stress resilience observed with NPY, indicating the importance of Ih in stress-related behavior.SIGNIFICANCE STATEMENT Resilience improves mental health outcomes in response to adverse situations. Neuropeptide Y (NPY) is associated with decreased stress responses and the expression of resilience in rodents and humans. Single or repeated injections of NPY into the basolateral amygdala (BLA) buffer negative behavioral effects of stress and induce resilience in rats, respectively. Here, we demonstrate that repeated administration of NPY into the BLA unfolds several cellular mechanisms that decrease the activity of pyramidal output neurons. One key mechanism is a reduction in levels of the excitatory ion channel HCN1. Moreover, shRNA knock-down of HCN1 expression in BLA recapitulates some of the actions of NPY and causes potent resilience to stress, indicating that this channel may be a possible target for therapy.

Therapeutic correction of ApoER2 splicing in Alzheimer's disease mice using antisense oligonucleotides.

Apolipoprotein E receptor 2 (ApoER2) is an apolipoprotein E receptor involved in long-term potentiation, learning, and memory. Given its role in cognition and its association with the Alzheimer's disease (AD) risk gene, apoE, ApoER2 has been proposed to be involved in AD, though a role for the receptor in the disease is not clear. ApoER2 signaling requires amino acids encoded by alternatively spliced exon 19. Here, we report that the balance of ApoER2 exon 19 splicing is deregulated in postmortem brain tissue from AD patients and in a transgenic mouse model of AD To test the role of deregulated ApoER2 splicing in AD, we designed an antisense oligonucleotide (ASO) that increases exon 19 splicing. Treatment of AD mice with a single dose of ASO corrected ApoER2 splicing for up to 6 months and improved synaptic function and learning and memory. These results reveal an association between ApoER2 isoform expression and AD, and provide preclinical evidence for the utility of ASOs as a therapeutic approach to mitigate Alzheimer's disease symptoms by improving ApoER2 exon 19 splicing.

Identification and characterization of Aß peptide interactors in Alzheimer's disease by structural approaches.

Currently, there are very limited pharmaceutical interventions for Alzheimer's disease (AD) to alleviate the amyloid burden implicated in the pathophysiology of the disease. Alzheimer's disease is characterized immunohistologically by the accumulation of senile plaques in the brain with afflicted patients progressively losing short-term memory and, ultimately, cognition. Although significant improvements in clinical diagnosis and care for AD patients have been made, effective treatments for this devastating disease remain elusive. A key component of the amyloid burden of AD comes from accumulation of the amyloid-beta (Aß) peptide which comes from processing of the amyloid precursor protein (APP) by enzymes termed secretases, leading to production of these toxic Aß peptides of 40-42 amino acids. New therapeutic approaches for reducing Aß are warranted after the most logical avenues of inhibiting secretase activity appear less than optimal in ameliorating the progression of AD.Novel therapeutics may be gleaned from proteomics biomarker initiatives to yield detailed molecular interactions of enzymes and their potential substrates. Explicating the APPome by deciphering protein complexes forming in cells is a complementary approach to unveil novel molecular interactions with the amyloidogenic peptide precursor to both understand the biology and develop potential upstream drug targets. Utilizing these strategies we have identified EC 3.4.24.15 (EP24.15), a zinc metalloprotease related to neprilysin (NEP), with the ability to catabolize Aß 1-42 by examining first potential in silico docking and then verification by mass spectrometry. In addition, a hormone carrier protein, transthyreitin (TTR), was identified and with its abundance in cerebrospinal fluid (CSF), found to clear Aß by inhibiting formation of oligomeric forms of Aß peptide. The confluence of complementary strategies may allow new therapeutic avenues as well as biomarkers for AD that will aid in diagnosis, prognosis and treatment.

Amyloid-beta and Alzheimer's disease: the role of neprilysin-2 in amyloid-beta clearance.

Accumulation of the amyloid-beta (Aß) peptide is a central factor in Alzheimer's disease (AD) pathogenesis as supported by continuing evidence. This review concisely summarizes this evidence supporting a critical role for Aß in AD before discussing the clearance of this peptide. Mechanisms of clearance of Aß are critical for preventing pathological elevations in Aß concentration. Direct degradation of Aß by endopeptidases has emerged as one important pathway for clearance. Of particular interest are endopeptidases that are sensitive to the neprilysin (NEP) inhibitors thiorphan and phosphoramidon (i.e., are "NEP-like") as these inhibitors induce a dramatic increase in Aß levels in rodents. This review will focus on neprilysin-2 (NEP2), a NEP-like endopeptidase which cooperates with NEP to control Aß levels in the brain. The evidence for the involvement of NEP2 in AD is discussed as well as the therapeutic relevance with regards to gene therapy and the development of molecular markers for the disease.

Of mice and men: neurogenesis, cognition and Alzheimer's disease.

Neural stem cells are maintained in the subgranular layer of the dentate gyrus and in the subventricular zone in the adult mammalian brain throughout life. Neurogenesis is continuous, but its extent is tightly regulated by environmental factors, behavior, hormonal state, age, and brain health. Increasing evidence supports a role for new neurons in cognitive function in rodents. Recent evidence delineates significant similarities and differences between adult neurogenesis in rodents and humans. Being context-dependent, neurogenesis in the human brain might be manifested differently than in the rodent brain. Decline in neurogenesis may play a role in cognitive deterioration, leading to the development of progressive learning and memory disorders, such as Alzheimer's disease. This review discusses the different observations concerning neurogenesis in the rodent and human brain, and their functional implications for the healthy and diseased brain.

Altered ryanodine receptor expression in mild cognitive impairment and Alzheimer's disease.

Intracellular Ca(2+) dysregulation is an underlying component of Alzheimer's disease (AD) pathophysiology, and recent evidence implicates the ryanodine receptor (RyR) in the disease pathway. Three genes code for different RyR isoforms and each gene transcript gives rise to several alternatively spliced messenger RNAs (mRNAs). These variants confer distinct functionality to the RyR channel, such as altering Ca(2+) release properties or subcellular localization. Changes in RyR isoform expression and alternative splicing have not been examined for potential roles in AD pathogenesis. Here, we compare mRNA levels of the RyR2 and RyR3 isoforms as well as specific alternatively spliced variants across vulnerable brain regions from postmortem samples of individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI), and AD. We find an increase in RyR2 transcripts in MCI brains compared with no cognitive impairment. In addition, there is a reduction in a RyR2 splice variant, associated with an antiapoptotic function, in MCI and AD brains. These alterations in RyR expression at early disease stages may reflect the onset of pathologic mechanisms leading to later neurodegeneration.

Altered NEP2 expression and activity in mild cognitive impairment and Alzheimer's disease.

Neprilysin-2 (NEP2), a close homolog of neprilysin (NEP), degrades amyloid-ß (Aß) and serves an important role in clearing Aß in vivo. We measured NEP2 and NEP mRNA levels from non-impaired (NI), mild cognitive impaired (MCI), and clinical Alzheimer's disease (AD) subjects in the mid-temporal gyrus, mid-frontal gyrus, caudate, and cerebellum. NEP2 activity levels were also determined. Our results indicate that NEP2 and NEP mRNA expression is altered in MCI subjects relative to NI subjects in AD-susceptible regions. NEP2 enzymatic activity was lowered in association with MCI and AD and was positively associated with cognitive function, independent of diagnostic category. Our finding that NEP2 expression and activity are altered in MCI is significant as these changes may potentially serve as preclinical markers for AD and reduced NEP2 activity may be associated with the development of AD.

Modification of pax6 and olig2 expression in adult hippocampal neurogenesis selectively induces stem cell fate and alters both neuronal and glial populations.

The generation of new neurons in the mammalian hippocampus continues throughout life, and lineage progression is regulated by transcription factors, local cues, and environmental influences. The ability to direct stem/progenitor cell fate in situ may have therapeutic potential. Using an in vivo retroviral delivery and lineage tracing approach, we compare the lineage-instruction factors, Pax6 and Olig2, and demonstrate that both participate in regulation of adult hippocampal neurogenesis in adult rats. We show that overexpression of the proneuronal factor Pax6 pushes neuronal precursor cells to early maturation and increases the frequency of neuronal phenotypes. However, Pax6 overexpression results in no net increase in neurogenesis at 3 weeks. Blocking of Olig2 function reduces and slows neuronal commitment and differentiation and decreases net neurogenesis. Altering expression of both factors also changes gliogenesis. Our results establish that Pax6 decreases the number of Neuron-Glia 2 progenitor cells and prevents oligodendrocytic lineage commitment, while repression of Olig2 results in an expanded astrocytic lineage. We conclude that selectively modifying transcriptional cues within hippocampal progenitor cells is sufficient to induce a cell fate switch, thus altering the neurogenesis-gliogenesis ratio. In addition, our data show the competence of multiple progenitor lineages to respond divergently to the same signal. Therefore, directing instructive cues to select phenotype and developmental stage could be critical to achieve precise outcomes in cell genesis. Further understanding the regulation of lineage progression in all progenitor populations within the target region will be important for developing therapeutic strategies to direct cell fate for brain repair.

Peripheral delivery of a CNS targeted, metalo-protease reduces aß toxicity in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD), an incurable, progressive neurodegenerative disorder, is the most common form of dementia. Therapeutic options have been elusive due to the inability to deliver proteins across the blood-brain barrier (BBB). In order to improve the therapeutic potential for AD, we utilized a promising new approach for delivery of proteins across the BBB. We generated a lentivirus vector expressing the amyloid ß-degrading enzyme, neprilysin, fused to the ApoB transport domain and delivered this by intra-peritoneal injection to amyloid protein precursor (APP) transgenic model of AD. Treated mice had reduced levels of Aß, reduced plaques and increased synaptic density in the CNS. Furthermore, mice treated with the neprilysin targeting the CNS had a reversal of memory deficits. Thus, the addition of the ApoB transport domain to the secreted neprilysin generated a non-invasive therapeutic approach that may be a potential treatment in patients with AD.

Neprilysin-2 is an important ß-amyloid degrading enzyme.

Proteases that degrade the amyloid-ß peptide (Aß) are important in protecting against Alzheimer's disease (AD), and understanding these proteases is critical to understanding AD pathology. Endopeptidases sensitive to inhibition by thiorphan and phosphoramidon are especially important, because these inhibitors induce dramatic Aß accumulation (∼30- to 50-fold) and pathological deposition in rodents. The Aß-degrading enzyme neprilysin (NEP) is the best known target of these inhibitors. However, genetic ablation of NEP results in only modest increases (∼1.5- to 2-fold) in Aß, indicating that other thiorphan/phosphoramidon-sensitive endopeptidases are at work. Of particular interest is the NEP homolog neprilysin 2 (NEP2), which is thiorphan/phosphoramidon-sensitive and degrades Aß. We investigated the role of NEP2 in Aß degradation in vivo through the use of gene knockout and transgenic mice. Mice deficient for the NEP2 gene showed significant elevations in total Aß species in the hippocampus and brainstem/diencephalon (∼1.5-fold). Increases in Aß accumulation were more dramatic in NEP2 knockout mice crossbred with APP transgenic mice. In NEP/NEP2 double-knockout mice, Aß levels were marginally increased (∼1.5- to 2-fold), compared with NEP(-/-)/NEP2(+/+) controls. Treatment of these double-knockout mice with phosphoramidon resulted in elevations of Aß, suggesting that yet other NEP-like Aß-degrading endopeptidases are contributing to Aß catabolism.

Intranasal phosphoramidon increases beta-amyloid levels in wild-type and NEP/NEP2-deficient mice.

Intranasal administration is emerging as a reliable and non-invasive method to bypass the blood-brain barrier and deliver drugs to the brain. This approach has been primarily used to explore therapeutic avenues for neurological diseases. However, intranasal administration could also be used to create animal models of brain disease. Beta-amyloid peptide (Aß) accumulation is a key feature of Alzheimer's disease (AD), and the most common models of AD are transgenic mice expressing mutant human genes linked to familial AD. An alternative model of amyloidosis utilizes intracerebroventricular infusion of thiorphan or phosphoramidon to block the activity of key Aß degrading enzymes (NEP, NEP2) resulting in accumulation of Aß. Here, we demonstrate that intranasal administration of phosphoramidon produces significantly elevated cerebral Aß levels in wild-type mice. Furthermore, intranasal phosphoramidon administration in double knockout mice lacking NEP and NEP2 also showed increased levels of Aß(40). These data show that intranasal delivery of drugs can be used to model AD and suggest that other phosphoramidon-sensitive peptidases are degrading Aß in NEP/NEP2-deficient mice.

Insights into neurogenesis and aging: potential therapy for degenerative disease?

Neurogenesis is the process by which new neural cells are generated from a small population of multipotent stem cells in the adult CNS. This natural generation of new cells is limited in its regenerative capabilities and also declines with age. The use of stem cells in the treatment of neurodegenerative disease may hold great potential; however, the age-related incidence of many CNS diseases coincides with reduced neurogenesis. This review concisely summarizes current knowledge related to adult neurogenesis and its alteration with aging and examines the feasibility of using stem cell and gene therapies to combat diseases of the CNS with advancing age.

Neurogenesis and Alzheimer's disease: at the crossroads.

While a massive and progressive neuronal loss in specific areas such as the hippocampus and cortex unequivocally underlies cognitive deterioration and memory loss in Alzheimer's disease, noteworthy alterations take place in the neurogenic microenvironments, namely, the subgranule layer of the dentate gyrus and the subventricular zone. Compromised neurogenesis presumably takes place earlier than onset of hallmark lesions or neuronal loss, and may play a role in the initiation and progression of neuropathology in Alzheimer's disease. Neurogenesis in the adult brain is thought to play a role in numerous forms and aspects of learning and memory and contribute to the plasticity of the hippocampus and olfactory system. Misregulated or impaired neurogenesis on the other hand, may compromise plasticity and neuronal function in these areas and exacerbate neuronal vulnerability. Interestingly, increasing evidence suggests that molecular players in Alzheimer's disease, including PS1, APP and its metabolites, play a role in adult neurogenesis. In addition, recent studies suggest that alterations in tau phosphorylation are pronounced in neurogenic areas, and may interfere with the potential central role of tau proteins in neuronal maturation and differentiation. On the other hand, numerous neurogenic players, such as Notch-1, ErbB4 and L1 are substrates of alpha- beta- and gamma- secretase that play a major role in Alzheimer's disease. This review will discuss current knowledge concerning alterations of neurogenesis in Alzheimer's disease with specific emphasis on the cross-talk between signaling molecules involved in both processes, and the ways by which familial Alzheimer's disease-linked dysfunction of these signaling molecules affect neurogenesis in the adult brain.