Amyotrophic lateral sclerosis damages proteostasis, affecting spinal and upper motor neurons earlier than a subset of cranial motor neurons. To aid disease understanding, we exposed induced cranial and spinal motor neurons (iCrMNs and iSpMNs) to proteotoxic stress, under which iCrMNs showed superior survival, quantifying the transcriptome and proteome for >8,200 genes at 0, 12, and 36 h. Two-thirds of the proteome showed cell-type differences. iSpMN-enriched proteins related to DNA/RNA metabolism, and iCrMN-enriched proteins acted in the endoplasmic reticulum (ER)/ER chaperone complex, tRNA aminoacylation, mitochondria, and the plasma/synaptic membrane, suggesting that iCrMNs expressed higher levels of proteins supporting proteostasis and neuronal function. When investigating the increased proteasome levels in iCrMNs, we showed that the activity of the 26S proteasome, but not of the 20S proteasome, was higher in iCrMNs than in iSpMNs, even after a stress-induced decrease. We identified Ublcp1 as an iCrMN-specific regulator of the nuclear 26S activity.

Amyotrophic Lateral Sclerosis (ALS) is a fatal adult-onset neurodegenerative disease without cure or understanding of its molecular basis. It is characterized by progressive loss of spinal and upper motor neurons, but surprising resistance of cranial motor neurons until the late stages of the disease. There is no known single-gene cause of ALS, suggesting a failure of the entire cellular system to maintain proteostasis. To generate new hypotheses on the molecular differences between cranial and spinal motor neurons that underlie ALS-sensitivity, we exploited a unique platform that generates highly pure populations of induced cranial and spinal motor neurons (iCrMN and iSpMN). Exposing both iCrMNs and iSpMNs to proteotoxic stress, we monitored transcriptome and proteome changes over 36 hours and identified significant hundreds of statistically significant differences at either or both the RNA and protein level from a core set of 8,206 genes with complete data. While we observed a response to misfolding stress coordinated between transcript and protein changes, differences between the cell type were specific to RNA and protein expression. iCrMNs and iSpMNs differed in expression of many membrane proteins, including synaptic proteins, solute carriers, channels, and signaling molecules, as well as genes involved in ribosome biogenesis and subunits of the core proteasome. Differences exclusive to the transcriptome involved chaperones and the tricarboxylic acid cycle. Further, we found that the superior proteostasis capability of iCrMNs might, in part, be caused due to higher activity and abundance of the fully assembled 26S/30S proteasome in the nucleus, pointing to an increased role of ubiquitin-mediated, targeted protein degradation. The formation of the 26S/30S proteasome was highly dynamic under stress, likely mediated by a surplus of regulatory particles that attached and detached based on the action of proteasome regulators. Several lines of evidence supported this notion and described an intricate network of differentially expressed proteasome regulators and ubiquitination enzymes that defined the iCrMNand iSpMN-specific ability to maintain proteostasis.

Despite clear therapeutic potential, the mechanisms that confer differential neuronal sensitivity are not well understood. During Amyotrophic Lateral Sclerosis (ALS), sensitive spinal motor neurons (SpMN) die while a subset of rostral cranial motor neurons (CrMN) survive. In this work, we optimized a protocol to differentiate CrMNs and SpMNs from human induced pluripotent stem cells (iPSCs) by direct programming and positional patterning. Human iCrMNs are more resistant than iSpMNs to proteotoxic stress and rely on the proteasome to maintain proteostasis. iCrMNs better prevent mislocalization of TDP43 from the nucleus, a hallmark of ALS progression. iSpMNs contain more splicing defects than iCrMNs in response to ALS-related stress with genes involved in splicing and proteostasis maintenance. Therefore, iCrMNs resist ALS at two levels, preventing protein accumulation and reducing splicing defects in response to TDP43 nuclear depletion. Thus, ALS-sensitive iSpMNs appear to enter a downward spiral compromising their ability to maintain proteostasis and splicing.

Amyotrophic Lateral Sclerosis (ALS) is a fatal adult neurodegenerative disease characterized by proteostasis dysregulation, resulting in progressive loss of spinal and upper motor neurons. A subset of cranial motor neurons resistant to ALS-stress survive until late stages of the disease. To investigate these differences, we exploited a unique platform of induced cranial and spinal motor neurons (iCrMNs and iSpMNs, respectively). Exposing both cell types to proteotoxic stress, we quantified transcriptome and proteome changes over 36 hours for a core set of >8,200 genes. While mRNA and protein changes under stress were congruent for many genes, cell-type specific differences manifested at either the RNA or protein level, but less at both. At the protein level, iCrMNs and iSpMNs differed significantly with respect to abundance of many membrane proteins, including synaptic proteins, solute carriers, adhesion molecules, and signaling molecules suggesting that the superior stress survival of iCrMNs involve diverse pathways supporting neuronal function. Other differences included genes involved in ribosome biogenesis and subunits of the core proteasome. We investigated the role of proteasomal degradation in more detail. Our data showed that although stress reduces proteasome activity in both neuronal types, iCrMNs had significantly more abundant and active 26S proteasome than iSpMNs, which indicate a higher capacity for the degradation of ubiquitinated proteins. We identified a new regulator of this better performance, i.e. the nuclear proteasome activator Ublcp1, whose inhibition sensitized iCrMNs, but not iSpMNs, to stress and abolished their higher survival rates. The results suggest that the two neuronal cell types regulate and use the degradation machinery differently under normal and stress conditions. Overall, this work demonstrates the value of unbiased system-wide analyses in generating hypotheses on differential proteostasis regulation in cranial and spinal motor neurons.

Amyotrophic Lateral Sclerosis (ALS) is an adult‐onset fatal neurodegenerative disease with a selective, highly progressive loss of both spinal and upper motor neurons. However, cranial motor neurons are much less sensitive and survive until the late stages of the disease. The inability of the cells to respond appropriately to Endoplasmic Reticulum (ER) stress is thought to be one of the major reasons for the ALS progression. ER stress is a condition that arises after the accumulation of misfolded proteins inside the ER and activates a repertoire of transcriptional and translational events collectively called unfolded protein response (UPR). In the context of ALS progression, the dynamics of transcriptomics and proteomic profile over time in the presence of ER stress conditions are poorly understood. Here, we established a condition where ER stress shows differential vulnerability of stem cell derived cranial (CrMN) and spinal motor (SpMN) neurons. Using high throughput quantitative omics approaches, we quantified >8,200 genes over time and found that subtle expression changes influence several pathways that are essential for cranial and spinal motor neuron function. We demonstrate that pathways related to overall protein turnover differ in these two cell lines. Specifically, we identified expression differences in Rab proteins that have not been recognized to‐date. These Rab proteins affect synaptic recycling, endocytic sorting, and autophagic flux. Further, we discovered a novel link between the core proteasome and endocytic pathways that potentially help CrMN to turn over proteins more efficiently than SpMN. Combined, these results illustrate new insights into the complex behavior and subtle differences between two motor neuron cell lines and provide new avenues towards therapeutic intervention.Support or Funding InformationNational Institute of General Medical Sciences (R01 GM113237), National Institute of General Medical Sciences (1R35GM127089‐01), Project ALS (A13‐0416)

The rise of single-cell transcriptomics has created an urgent need for similar approaches that use a minimal number of cells to quantify expression levels of proteins. We integrated and optimized multiple recent developments to establish a proteomics workflow to quantify proteins from as few as 1000 mammalian stem cells. The method uses chemical peptide labeling, does not require specific equipment other than cell lysis tools, and quantifies >2500 proteins with high reproducibility. We validated the method by comparing mouse embryonic stem cells and in vitro differentiated motor neurons. We identify differentially expressed proteins with small fold changes and a dynamic range in abundance similar to that of standard methods. Protein abundance measurements obtained with our protocol compared well to corresponding transcript abundance and to measurements using standard inputs. The protocol is also applicable to other systems, such as fluorescence-activated cell sorting (FACS)-purified cells from the tunicate Ciona. Therefore, we offer a straightforward and accurate method to acquire proteomics data from minimal input samples.

The rise of single-cell transcriptomics has created an urgent need for similar approaches that use a minimal number of cells to quantify expression levels of proteins. We integrated and optimized multiple recent developments to establish a proteomics workflow to quantify proteins from as few as 1,000 mammalian stem cells. The method uses chemical peptide labeling, does not require specific equipment other than cell lysis tools, and quantifies >2,500 proteins with high reproducibility. We validated the method by comparing mouse embryonic stem cells and in vitro differentiated motor neurons. We identify differentially expressed proteins with small fold-changes, and a dynamic range in abundance similar to that of standard methods. Protein abundance measurements obtained with our protocol compare well to corresponding transcript abundance and to measurements using standard inputs. The protocol is also applicable to other systems, such as FACS-purified cells from the tunicate Ciona . Therefore, we offer a straightforward and accurate method to acquire proteomics data from minimal input samples.

In amyotrophic lateral sclerosis (ALS) spinal motor neurons (SpMN) progressively degenerate while a subset of cranial motor neurons (CrMN) are spared until late stages of the disease. Using a rapid and efficient protocol to differentiate mouse embryonic stem cells (ESC) to SpMNs and CrMNs, we now report that ESC-derived CrMNs accumulate less human (h)SOD1 and insoluble p62 than SpMNs over time. ESC-derived CrMNs have higher proteasome activity to degrade misfolded proteins and are intrinsically more resistant to chemically-induced proteostatic stress than SpMNs. Chemical and genetic activation of the proteasome rescues SpMN sensitivity to proteostatic stress. In agreement, the hSOD1 G93A mouse model reveals that ALS-resistant CrMNs accumulate less insoluble hSOD1 and p62-containing inclusions than SpMNs. Primary-derived ALS-resistant CrMNs are also more resistant than SpMNs to proteostatic stress. Thus, an ESC-based platform has identified a superior capacity to maintain a healthy proteome as a possible mechanism to resist ALS-induced neurodegeneration.

Aging is accompanied by declines in memory performance, and particularly affects memories that rely on hippocampal-cortical systems, such as episodic and explicit. With aged populations significantly increasing, the need for preventing or rescuing memory deficits is pressing. However, effective treatments are lacking. Here, we show that the level of the mature form of insulin-like growth factor 2 (IGF-2), a peptide regulated in the hippocampus by learning, required for memory consolidation and a promoter of memory enhancement in young adult rodents, is significantly reduced in hippocampal synapses of aged rats. By contrast, the hippocampal level of the immature form proIGF-2 is increased, suggesting an aging-related deficit in IGF-2 processing. In agreement, aged compared to young adult rats are deficient in the activity of proprotein convertase 2, an enzyme that likely mediates IGF-2 posttranslational processing. Hippocampal administration of the recombinant, mature form of IGF-2 rescues hippocampal-dependent memory deficits and working memory impairment in aged rats. Thus, IGF-2 may represent a novel therapeutic avenue for preventing or reversing aging-related cognitive impairments.